Methods of generating and expanding hematopoietic stem cells

ABSTRACT

Production and maintenance of hematopoietic stem cells in in vitro culture systems has proven to be elusive. Disclosed herein are hematopoietic stem cell compositions that originate from organoids such as liver organoids. These organoids also comprise a rare type of immune cell that is not yet fully elucidated due to the difficulty in isolating said immune cell from biological samples. Also disclosed herein are methods of producing said hematopoietic stem cells and immune cells from organoids, as well as methods of expanding hematopoietic stem cells from other sources using these organoids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalPatent Application No. 62/855,524, filed May 31, 2019 and U.S.Provisional Patent Application No. 62/855,536, filed May 31, 2019, eachof which is hereby expressly incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledCHMC63_019WOSeqListing.TXT, which was created and last modified on May18, 2020, which is 2,077 bytes in size. The information in theelectronic Sequence Listing is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

Aspects of the present disclosure relate generally to hematopoietic stemcell compositions and methods of making thereof.

BACKGROUND

Clinically, hematopoietic stem cell (HSC) transplants derived from bonemarrow are common procedures performed more than 40,000 times a yearworldwide. These transplants can be done to treat various types of bloodcancers, immune disorders, and gene defects in blood cells. HSCtransplants can be performed either autologously (using the patient'sown cells) or allogeneically (using a human leukocyte antigen (HLA)matched donor). However, autologous transplants are often not an optiondue to limited availability of the patient's own healthy cells andallogeneic transplants can result in graft vs. host disease (GVHD) thatcan be chronic and require immunosuppressants that put the patient atrisk for infection. Because of this, allogeneic transplants have a lowersurvival rate.

While HSCs from umbilical cord blood (UCB) may be collected for use in apatient, the number of cells that can he obtained this way is limitedand there are currently no culture methods to significantly expand theirnumbers in vitro. Furthermore, previous attempts to produce HSCs fromiPSCs require introduction of foreign transgenes that pose unknownrisks, thus hampering potential clinical applications. There is apresent and lasting need for robust production and expansion of HSCs,fbr example for use in autologous or allogeneic transplants.

SUMMARY

Some aspects of the present disclosure relate to methods of producing aliver organoid that produces hematopoietic stem cells. In someembodiments, the methods comprise culturing a foregut spheroid underconditions sufficient to differentiate the foregut spheroid into a liverorganoid. In some embodiments, the liver organoid comprises a CD34⁺hemogenic endothelium population that differentiate into hematopoieticstem cells. In some embodiments, culturing the foregut spheroid underconditions sufficient to differentiate the foregut spheroid into a liverorganoid comprises contacting the foregut spheroid with one or more(e.g. at least 1, 2, 3) of an FGF signaling pathway activator, a Wntsignaling pathway activator, or a BMP inhibitor, or any combinationthereof. In some embodiments, culturing the foregut spheroid underconditions sufficient to differentiate the foregut spheroid into a liverorganoid comprises contacting the foregut spheroid with an FGF signalingpathway activator or a Wnt signaling pathway activator, or both. In someembodiments, the FGF signaling pathway activator is FGF4. In someembodiments, the Wnt signaling pathway activator is CHIR99021. In someembodiments, the BMP inhibitor is Noggin. In some embodiments, culturingthe foregut spheroid comprises contacting the foregut spheroid with aNotch activator or ligand. In some embodiments, the Notch activator orligand is, comprises, consists essentially of, or consists of one ormore of DLL1, DLL3, DLL4, JAG1, or JAG2, or any combination thereof. Insome embodiments, the Notch activator or ligand is, comprises, consistsessentially of, or consists of a cell that expresses one or more ofDLL1, DLL3, JAG1, or JAG2, or any combination thereof. In someembodiments, the foregut spheroid comprises a cell that expresses one ormore Notch activators or ligands selected from the group consisting ofDLL1, DLL3, DLL4, JAG1, or JAG2, or any combination thereof. In someembodiments, culturing the foregut spheroid comprises contacting theforegut spheroid with one or more of IL-3, IL-34, or GM-CSF, or anycombination thereof. In sonic embodiments, culturing the foregutspheroid comprises contacting the foregut spheroid with stem cell factor(SCF) or thrombopoietin (TPO), or both. In some embodiments, culturingthe foregut spheroid does not comprise contacting the foregut spheroidwith retinoic acid (RA). In some embodiments, culturing the foregutspheroid does not comprise contacting the foregut spheroid with one ormore of dexamethasone, Oncostatin M, or hepatocyte growth factor, or anycombination thereof In some embodiments, the hematopoietic stem cellsare not exogenously expressing one or more of ERG, HOXA5, HOXA9, HOXA10,LCOR, RUNX1, SPI1, FOSB, or GFI1, or any combination thereof. In someembodiments, the methods comprise isolating the hemogenic endotheliumpopulation or the hematopoietic stem cells, or both, from the liverorganoid. In some embodiments, isolating the hemogenic endotheliumpopulation or the hematopoietic stem cells, or both, is performed bysorting for a cell population from the liver organoid that is CD34+,CD45+, or both. In some embodiments, the methods comprise culturinginduced pluripotent stem cells under conditions sufficient todifferentiate the induced pluripotent stem cells into definitiveendoderm and culturing the definitive endoderm under conditionssufficient to differentiate the definitive endoderm into the foregutspheroid prior to culturing the foregut spheroid under conditionssufficient to differentiate the foregut spheroid into the liverorganoid. In some embodiments, the methods comprise culturing adefinitive endoderm under conditions sufficient to differentiate thedefinitive endoderm into the foregut spheroid prior to culturing theforegut spheroid under conditions sufficient to differentiate theforegut spheroid into the liver organoid.

Some aspects of the present disclosure relate to liver organoids. Insome embodiments, the liver organoids are the liver organoids producedby any one of the methods disclosed herein.

Some aspects of the present disclosure relate to a hemogenic endotheliumpopulation, In some embodiments, the hemogenic endothelium population isproduced by any one of the methods disclosed herein.

Some aspects of the present disclosure relate to hematopoietic stemcells. in some embodiments, the hematopoietic stem cells are produced byany one of the methods disclosed herein.

Some aspects of the present disclosure relate to methods for isolatinglymphoid cells from a liver organoid. In some embodiments, the liverorganoid is any one of the liver organoids produced by any one of themethods disclosed herein. In some embodiments, the lymphoid cells areisolated from any one of the liver organoids produced by any one of themethods described herein. In some embodiments, the lymphoid cellscomprise one or more of B cells, T cells, NK cells, or common lymphoidprogenitor cells. In some embodiments, the B cells are B1 cells. In someembodiments, the B cells are B2 cells. In some embodiments, the B cellsare both B1 cells and B2 cells. In some embodiments, prior to isolatingthe lymphoid cells, the methods comprise making a single cell suspensionfrom the liver organoid and culturing the single cell suspension onstromal cells. In some embodiments, culturing the single cell suspensionon stromal cells expands lymphoid cells in the single cell suspension.In some embodiments, the stromal cells are MS-5 cells. In someembodiments, the single cell suspension is contacted with one or more ofFLT3 ligand, SCF, or IL-7, or any combination thereof. In someembodiments, the B1 cells are CD20⁺CD43⁺CD27⁺. In some embodiments,isolating the B1 cells is performed by sorting for a cell populationfrom the single cell suspension that is one or more of CD20⁺, CD43+,CD27⁺, or any combination thereof.

Some aspects of the present disclosure relate to lymphoid cells. In someembodiments, the lymphoid cells are the lymphoid cells produced by anyone of the methods described herein. In some embodiments, the lymphoidcells are B1 cells, and the B1 cells are the B1 cells produced by anyone of the methods described herein.

Some aspects of the present disclosure relate to methods of expanding apopulation of hematopoietic stem cells. In some embodiments, the methodscomprise culturing the population of hematopoietic stem cells in contactwith a liver organoid. In some embodiments, the liver organoid comprisesa hematopoietic niche environment. In some embodiments, the liverorganoid is the liver organoid produced by any one of the methodsdescribed herein. In some embodiments, culturing the population ofhematopoietic stem cells comprises contacting the population ofhematopoietic stem cells with SCF or TPO, or both. In some embodiments,the expanded population of hematopoietic stem cells maintainpluripotency. In some embodiments, the population of hematopoietic stemcells is obtained from umbilical cord blood, mobilized peripheral blood,bone marrow, pluripotent stem cells, embryonic stem cells, fetal liver,or fetal spleen, or any combination thereof. In some embodiments, thepopulation of hematopoietic stem cells is autologous or allogeneic tothe liver organoid. In some embodiments, the population of hematopoieticstem cells is obtained from an individual. In some embodiments, theindividual is an individual in need of hematopoietic stem cells. In someembodiments, the liver organoid is produced from induced pluripotentstem cells derived from the individual.

Some aspects of the present disclosure relate to methods ofadministering to an individual in need a population of hematopoieticstem cells. In some embodiments, the population of hematopoietic stemcells is the population of hematopoietic stem cells produced by any oneof the methods described herein. In some embodiments, the population ofhematopoietic stem cells is autologous or allogeneic to the individualin need.

Some aspects of the present disclosure relate to a cell composition. Insome embodiments, the cell composition comprises a population ofhematopoietic stem cells and a liver organoid. In some embodiments, thepopulation of hematopoietic stem cells is the population ofhematopoietic stem cells produced by any one of the methods describedherein. In some embodiments, the liver organoid is the liver organoidproduced by any one of the methods described herein. In someembodiments, the population of hematopoietic stem cells and liverorganoid are from the same individual. In some embodiments, thepopulation of hematopoietic stem cells and liver organoid are fromdifferent individuals.

Some aspects of the present disclosure relate to methods of treating anindividual in need. In some embodiments, the methods compriseadministering to the individual in need an isolated heinogenicendothelium population. In some embodiments, the isolated hemogenicendothelium population is the isolated hemogenic endothelium populationproduced by any one of the methods described herein. In someembodiments, the methods comprise administering to the individual inneed isolated hematopoietic stem cells. In some embodiments, theisolated hematopoietic stem cells are the isolated hematopoietic stemcells produced by any one of the methods described herein. In someembodiments, the methods comprise administering to the individual inneed isolated lymphoid cells. In some embodiments, the isolated lymphoidcells are the isolated lymphoid cells are produced by any one of themethods described herein. In some embodiments, the methods compriseadministering to the individual in need isolated. B1 cells. In someembodiments, the isolated B1 cells are the isolated B1 cells produced byany one of the methods described herein.

Embodiments of the present disclosure provided herein are described byway of the following numbered alternatives:

1. A method for generating hematopoietic cells in a liver organoid,comprising contacting a foregut spheroid an FGF signaling pathwayactivator, a Wnt signaling pathway activator, and a BMP inhibitor toform a liver organoid having a mesoderm cell population;

wherein said liver organoid produces hematopoietic stem cells (HSC).

2. The method of alternative I, wherein said foregut spheroid comprisesmesenchyme.

3. The method of alternative 1 or 2, comprising contacting said foregutspheroid with a Notch activator.

4. The method of alternative 3, wherein said Notch activator is selectedfrom one or more proteins selected from DLI1, DLL3, DLL4, JAG-1, JAG2,and combinations thereof.

5. The method of alternative 3, wherein said Notch activator is selectedfrom one or more cells expressing DLL1, DLL3, DLL4, JAG1, JAG2, andcombinations thereof

6. The method of any preceding alternative, wherein said Notch activatoris a bone marrow cell line expressing one or more of DLL1, DLL3, DLL4,JAG1, JAG2, and combinations thereof.

7. The method of any preceding alternative, comprising the step ofcontacting said liver organoid with a cytokine.

8. The method of alternative 7, wherein said cytokine is added at aconcentration of about 1 to about 10 ng/mL, from about 2 to from about15 ng/mL, from about 3 to from about 20 ng/mL, from about 4 to fromabout 25 ng/mL, or from about 5 to from about 30 ng/mL.

9. The method of alternative 1, wherein said HLO expresses one or moreof albumin (gene accession number HGNC:399), alpha-fetoprotein (HGNC:317), and erythropoietin (HGNC: 3415).

10. The method of any preceding alternatives, wherein said hematopoieticstem cells (HSC) are identified by flow cytometry as one or more oflin-, CD34+, CD38−, CD90+, CD45RA−, CD49f+.

11. The method of any preceding alternative, wherein said hetnatopoieticstem cells are at a concentration of about 1/10,000 cells in HLOculture.

12. The method of alternative 3, wherein said Notch activator activatesNotch 4 (HC NC: 7884).

13. A method for expanding a population of stem cells, comprisingculturing said stem cells in the presence of a liver organoid.

14. The method of alternative 13, wherein said stem cells are obtainedfrom umbilical cord blood, peripheral blood, fetal liver, fetal spleen,bone marrow, gut, yolk sac, or combinations thereof.

15. The method of alternative 13 or 14, wherein said expanded stem cellsmaintain pluripotency.

16. The method of any preceding alternative wherein said stem cells areobtained from an individual in need of treatment with stem cells.

17. The method of any preceding alternative comprising administeringsaid expanded stem cells to an individual in need thereof.

18. The method of any preceding alternative comprising geneticallymodifying said stem cells one or more time periods selected from beforeexpansion, during expansion, and after expansion.

BRIEF DESCRIPTION OF THE DRAWINGS

In addition to the features described above, additional features andvariations will be readily apparent from the following descriptions ofthe drawings and exemplary embodiments. It is to be understood thatthese drawings depict embodiments and are not intended to be limiting inscope.

FIG. 1A depicts an embodiment of a schematic for liver organoiddifferentiation methods.

FIG. 1B depicts an embodiment of histology of day 18 liver organoidcultures in Matrigel drops with hematoxylin/eosin andimmunohistochemistry (IHC) for albumin, alpha-fetoprotein, CD34 andCD45. Boxed regions are shown as zoomed images in bottom right inset.

FIG. 1C depicts an embodiment of expression of CD34, AFP, HBG1, and ALBin liver organoid cultures of different days as measured by rt-PCR.

FIG. 1D depicts an embodiment of albumin expression by liver organoidcultures cultured with liver differentiation media (RA/HCM) orundifferentiated liver organoid (+FGF+BMP/HCM and +FGF+BMP) culturesfrom different iPSC lines (72_3, 1383D6, 449) as measured by ELISA.

FIG. 2 depicts an embodiment of a t-SNE plot of scRINA-set data from day14 organoid cultures with different populations of interest labeled.

FIG. 3 depicts an embodiment of a t-SNE plot of snRNA-seq data from day14 organoid cultures with liver, endothelial/HSC, and erythroidpopulations labeled.

FIGS. 4A-C depict an embodiment of t-SNE plots of relevant genesindicating (A) a hepatic population, (B) a hemogenic endothelialpopulation, and (C) an erythroid population.

FIG. 5A depicts an embodiment of representative images from Giemsastains after CFC assays performed from cells of day 14 organoidcultures.

FIG. 5B depicts an embodiment of an assay of colony forming units fromCD34⁺ UCB cells, day 18 liver organoid cells, and undifferentiatediPSCs.

FIG. 6A depicts an embodiment of immunofluorescence images showing thepresence of liver organoid cells stained for EpCAM, endothelial cellsstained for CD34, and an adjacent cluster of erythrocytes stained forGYPA.

FIG. 6B depicts an embodiment of IHC staining of CD34 and CD45 in day 18organoid cultures.

FIG. 6C depicts an embodiment of flow cytometry plots detecting CD34 inorganoid cultures at timepoints day 11, 13, and 15.

FIG. 6D depicts an embodiment of a graph showing number of coloniesformed from cells of organoid cultures of different days ofdifferentiation compared to UCB CD34⁺ control.

FIG. 6E depicts an embodiment of a graph showing gPCR results indicatingexpression levels of HES1 at different days of organoid culture.

FIG. 7A depicts an embodiment of t-SNE plots of scRNA-seq data showing apopulation of cells co-expressing CD34 and several genes related toNotch signaling.

FIG. 7B depicts an embodiment of a schematic for preparing liverorganoids and addition of DLL4 at day 6 of culture.

FIG. 8 depicts an embodiment of CD45 expression in liver organoidcultures after addition of DLL4 and cytokines (IL-3, IL-34, GM-CSF).

FIG. 9A depicts an embodiment of a graph showing qPCR results of IL-6expression in organoids at day 18 (N=negative control, D=DLL4 added,C=cytokines added, DC=DLL4 and cytokines added).

FIG. 9B depicts an embodiment of a graph showing qPCR results of albuminexpression in organoids at day 18 (N=negative control, D=DLL4 added,C=cytokines added, DC=DLL4 and cytokines added).

FIG. 10A depicts an embodiment of light microscopy images of organoidcultures with either DMSO control or 100 μM DAPT after 12 days oftreatment.

FIG. 10B depicts an embodiment of a schematic for adding DAPT at day 6of culture for 4 days (removing DAPT at day 10 of culture).

FIG. 10C depicts an embodiment of HES1 expression at differentconcentrations of DAPT after 4 days of treatment.

FIG. 10D depicts an embodiment of ALB expression at differentconcentrations of DAPT after 4 days of treatment.

FIG. 11A depicts an embodiment of a schematic demonstrating a co-culturesystem layout. MS-5 cells are cultured with FLT3L, SCF, and IL-7.

FIG. 11B depicts an embodiment of brightfield images showing day 20 ofMS-5 co-culture with either UCB cells or organoid culture cells.

FIG. 11C depicts an embodiment of flow cytometry plots of day 26organoid/15-5 co-cultures. Leftmost panels indicate CD45 and CD20subsets that are further gated into CD43 and CD27 in the next panels.

FIG. 12A depicts an embodiment of histology of day 18 organoid culturesshowing IHC for CD34 and Nestin.

FIG. 12B depicts an embodiment of immunofluorescence images showingDAPI, CD34, and Nestin in day 18 organoid cultures.

FIG. 12C depicts an embodiment of t-SNE plots of scRNA-seq data showingexpression of TPO and SCF.

FIG. 12D depicts an embodiment of flow cytometry plots of CD34 and CD45in day 18 organoid cultures with and without the addition of SCF andTPO.

FIG. 12E depicts an embodiment of a graph showing the number of CFCcolonies produced per Matrigel drop at organoid culture day 18 after theaddition of UCB cells, SCF, and TPO at organoid culture day 6. “H+U”indicates HLO and UCB cells combined in the Matrigel drop.

FIG. 12F depicts an embodiment of a graph showing the number of CFCcolonies formed per 1000 UCB cells. X axis indicates organoid cultureday. UCB cells were combined with HLO at organoid culture day 6. “UCB”indicates fresh UCB cell control not co-cultured with HLO, “DMEM-RA”indicates UCB cells and HLO co-cultured in advanced DMEM alone (withoutretinoic acid). “HCM” indicates UCB cells and HLO co-cultured in 4 daysof DMEM+RA (organoid culture days 6-10) followed by HCM liverdifferentiation media.

FIG. 12G depicts an embodiment of a graph showing relative amounts ofdifferent colony types in CFC assays at different conditions shown inFIG. 12F.

FIG. 13A depicts an embodiment of a schematic demonstrating layout ofmouse engraftment experiments.

FIG. 13B depicts an embodiment of flow cytometry blots detecting CD45 inisolated PMBCs from mice 7 weeks after injecting cells.

DETAILED DESCRIPTION

Creation of hematopoietic stem cells (HSCs) from induced pluripotentstem cells (iPSCs) has great potential for clinical use yet has so farbeen elusive. Recent successes have been seen with the addition ofexogenous genes (e.g. ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, SPI1,FOSB, or GFI1) either to iPS cells or endothelial cells for directconversion. However, the genomic manipulations required for this featlimit any potential clinical translation. At the same time, the adventof organoid culture systems has recently provided researchers with apowerful tool to mimic complicated systems consisting of multiple celltypes and study them in a controlled setting over time. The organoidsystems disclosed herein recreates the complex niche of the fetal livermicroenvironment to provide a more efficient way of studyinghematopoiesis in vitro with the goal of creating cells for use in theclinic. Production of hematopoietic cells in organoid systems areexplored in PCT Publication WO 2020/056158.

Hematopoiesis in the fetal liver is a unique time window in which HSCsundergo massive expansion. Because of this, it is particularlyattractive as an HSC niche to model. HSCs are unable to maintain inculture and have thus far shown limited expansion in vitro, althoughsome recent success has been seen with mouse HSC using a cocktail ofgrowth factors. The current state of the art for in vitro HSC expansionimplements a system of media containing hematopoietic cytokines tomaintain and expand cells. However, this does not fully reproduce thecomplex microenvironment involving multiple cell types interactingdirectly and through transient signaling. The organoid systems describedherein provide a unique opportunity to model this niche in a way thatallows for expansion of HSCs for therapeutic use.

Modeling the fetal liver microenvironment allows for creation of B1cells, a unique type of hematopoietic cell that differentiates duringfetal development. These cells are made specifically in the fetal liverand exist in only very low levels after birth in the spleen andperitoneum. Due to their exceedingly small quantity in adults, most B1cell studies use mouse cells while B1 cells in humans have not yet beenfully described and potential progenitor populations are still unknown.Another aspect of B1 cells is their ability to secrete IgM without beingstimulated. Because of this, they are responsible for the initialresponse during vaccination. Additional analysis into theirdifferentiation and development may allow for their creation in vitroand provide a useful source of antibodies and aid in potential vaccinedevelopment.

The organoid systems described herein are positioned for great clinicalimpact. Cells from patients needing HSC transplants can have iPSCsgenerated that are then used to create customized hSCs that can bere-implanted without risk of rejection or GVHD, thus fulfilling the needfor a large supply of HSCs for use in autologous or allogeneictransplants.

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented herein. It will be readily understood that the aspects of thepresent disclosure, as generally described herein, and illustrated inthe Figures, can be arranged, substituted, combined, separated, anddesigned in a wide variety of different configurations, all of which areexplicitly contemplated herein.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood when read in light of theinstant disclosure by one of ordinary skill in the art to which thepresent disclosure belongs. For purposes of the present disclosure, thefollowing terms are explained below.

The embodiments herein are generally disclosed using affirmativelanguage to describe the numerous embodiments. Embodiments also includeones in which subject matter is excluded, in full or in part, such assubstances or materials, method steps and conditions, protocols, orprocedures.

The articles “a” and “an” are used herein to refer to one or to morethan one (for example, at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 10% to a reference quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length.

Throughout this specification, unless the context requires otherwise,the words “comprise,” “comprises,” and “comprising” will be understoodto imply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. By “consisting of” is meant including, and limitedto, whatever follows the phrase “consisting of.” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory, and that no other elements may be present. By “consistingessentially of” is meant including any elements listed after the phrase,and limited to other elements that do not interfere with or contributeto the activity or action specified in the disclosure for the listedelements. Thus, the phrase “consisting essentially of” indicates thatthe listed elements are required or mandatory, but that other elementsare optional and may or may not be present depending upon whether or notthey materially affect the activity or action of the listed elements.

The terms “individual”, “subject”, or “patient” as used herein havetheir plain and ordinary meaning as understood in light of thespecification, and mean a human or a non-human mammal, e.g., a dog, acat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate,or a bird, e.g., a chicken, as well as any other vertebrate orinvertebrate. The term “mammal” is used in its usual biological sense.Thus, it specifically includes, but is not limited to, primates,including simians (chimpanzees, apes, monkeys) and humans, cattle,horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice,guinea pigs, or the like.

The terms “effective amount” or “effective dose” as used herein havetheir plain and ordinary meaning as understood in light of thespecification, and refer to that amount of a recited composition orcompound that results in an observable effect. Actual dosage levels ofactive ingredients in an active composition of the presently disclosedsubject matter can be varied so as to administer an amount of the activecomposition or compound that is effective to achieve the desiredresponse for a particular subject and/or application. The selecteddosage level will depend upon a variety of factors including, but notlimited to, the activity of the composition, formulation, route ofadministration, combination with other drugs or treatments, severity ofthe condition being treated, and the physical condition and priormedical history of the subject being treated. In some embodiments, aminimal dose is administered, and dose is escalated in the absence ofdose-limiting toxicity to a minimally effective amount. Determinationand adjustment of an effective dose, as well as evaluation of when andhow to make such adjustments, are contemplated herein.

The terms “function” and “functional” as used herein have their plainand ordinary meaning as understood in light of the specification, andrefer to a biological, enzymatic, or therapeutic function.

The term “inhibit” as used herein has its plain and ordinary meaning asunderstood in light of the specification, and may refer to the reductionor prevention of a biological activity. The reduction can be by apercentage that is, is about, is at least, is at least about, is notmore than, or is not more than about, 10%, 20%, 30%, 40%. 50%, 60%, 70%,80%, 90%, or 100%, or an amount that is within a range defined by anytwo of the aforementioned values. As used herein, the term “delay” hasits plain and ordinary meaning as understood in light of thespecification, and refers to a slowing, postponement, or deferment of abiological event, to a time which is later than would otherwise beexpected. The delay can be a delay of a percentage that is, is about, isat least, is at least about, is not more than, or is not more thanabout, 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or anamount within a range defined by any two of the aforementioned values.The terms inhibit and delay may not necessarily indicate a 100%inhibition or delay. A partial inhibition or delay may be realized.

As used herein, the term “isolated” has its plain and ordinary meaningas understood in light of the specification, and refers to a substanceand/or entity that has been (1) separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature and/or in an experimental setting), and/or (2) produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from equal to, about, at least, atleast about, not more than, or not more than about, 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of theother components with which they were initially associated (or rangesincluding and/or spanning the aforementioned values). In someembodiments, isolated agents are, are about, are at least, are at leastabout, are not more than, or are not more than about, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, substantially 100%, or 100%pure (or ranges including and/or spanning the aforementioned values). Asused herein, a substance that is “isolated” may be “pure” (e.g.,substantially free of other components). As used herein, the term“isolated cell” may refer to a cell not contained in a multi-cellularorganism or tissue.

As used herein, “in vivo” is given its plain and ordinary meaning asunderstood in light of the specification and refers to the performanceof a method inside living organisms, usually animals, mammals, includinghumans, and plants, as opposed to a tissue extract or dead organism.

As used herein, “ex vivo” is given its plain and ordinary meaning asunderstood in light of the specification and refers to the performanceof a method outside a living organism with little alteration of naturalconditions.

As used herein, “in vitro” is given its plain and ordinary meaning asunderstood in light of the specification and refers to the performanceof a method outside of biological conditions, e.g., in a petri dish ortest tube.

The terms “nucleic acid” or “nucleic acid molecule” as used herein havetheir plain and ordinary meaning as understood in light of thespecification, and refer to polynucleotides, such as deoxyribonucleicacid (DNA) or ribonucleic acid (RNA), oligonucleotides, those thatappear in a cell naturally, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,enantiomeric forms of naturally-occurring nucleotides), or a combinationof both. Modified nucleotides can have alterations in sugar moietiesand/or in pyrimidine or purine base moieties. Sugar modificationsinclude, for example, replacement of one or more hydroxyl groups withhalogens, alkyl groups, amines, and azido groups, or sugars can befunctionalized as ethers or esters. Moreover, the entire sugar moietycan be replaced with sterically and electronically similar structures,such as aza-sugars and carbocyclic sugar analogs. Examples ofmodifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, or phosphoramidate. The term “nucleic acid molecule”also includes so-called “peptide nucleic acids,” which comprisenaturally-occurring or modified nucleic acid bases attached to apolyamide backbone. Nucleic acids can be either single stranded ordouble stranded. “Oligonucleotide” can be used interchangeable withnucleic acid and can refer to either double stranded or single strandedDNA or RNA. A nucleic acid or nucleic acids can be contained in anucleic acid vector or nucleic acid construct (e.g. plasmid, virus,retrovirus, lentivirus, bacteriophage, cosmid, fosmid, phagemid,bacterial artificial chromosome (BAC), yeast artificial chromosome(YAC), or human artificial chromosome (HAC)) that can be used foramplification and/or expression of the nucleic acid or nucleic acids invarious biological systems. Typically, the vector or construct will alsocontain elements including but not limited to promoters, enhancers,terminators, inducers, ribosome binding sites, translation initiationsites, start codons, stop codons, polyadenylation signals, origins ofreplication, cloning sites, multiple cloning sites, restriction enzymesites, epitopes, reporter genes, selection markers, antibiotic selectionmarkers, targeting sequences, peptide purification tags, or accessorygenes, or any combination thereof.

A nucleic acid or nucleic acid molecule can comprise one or moresequences encoding different peptides, polypeptides, or proteins. Theseone or more sequences can be joined in the same nucleic acid or nucleicacid molecule adjacently, or with extra nucleic acids in between, e.g.linkers, repeats or restriction enzyme sites, or any other sequence thatis, is about, is at least, is at least about, is not more than, or isnot more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 150, 200, or 300 bases long, or any length in a rangedefined by any two of the aforementioned lengths. The term “downstream”on a nucleic acid as used herein has its plain and ordinary meaning asunderstood in light of the specification and refers to a sequence beingafter the 3′-end of a previous sequence, on the strand containing theencoding sequence (sense strand) if the nucleic acid is double stranded.The term “upstream” on a nucleic acid as used herein has its plain andordinary meaning as understood in light of the specification and refersto a sequence being before the 5′-end of a subsequent sequence, on thestrand containing the encoding sequence (sense strand) if the nucleicacid is double stranded. The term “grouped” on a nucleic acid as usedherein has its plain and ordinary meaning as understood in light of thespecification and refers to two or more sequences that occur inproximity either directly or with extra nucleic acids in between, e.g.linkers, repeats, or restriction enzyme sites, or any other sequencethat is, is about, is at least, is at least about, is not more than, oris not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a rangedefined by any two of the aforementioned lengths, but generally not witha sequence in between that encodes for a functioning or catalyticpolypeptide, protein, or protein domain.

The nucleic acids described herein comprise nucleobases. Primary,canonical, natural, or unmodified bases are adenine, cytosine, guanine,thymine, and uracil. Other nucleobases include but are not limited topurines, pyrimidines, modified nucleobases, 5-methylcytosine,pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine,xanthine, 5,6-dihydrouracil, 5-hydroxymethyicytosine, 5-bromouracil,isoguanine, isocytosine, aminoallyl bases, dye-labeled bases,fluorescent bases, or biotin-labeled bases.

The terms “peptide”, “polypeptide”, and “protein” as used herein havetheir plain and ordinary meaning as understood in light of thespecification and refer to macromolecules comprised of amino acidslinked by peptide bonds. The numerous functions of peptides,polypeptides, and proteins are known in the art, and include but are notlimited to enzymes, structure, transport, defense, hormones, orsignaling. Peptides, polypeptides, and proteins are often, but notalways, produced biologically by a ribosomal complex using a nucleicacid template, although chemical syntheses are also available. Bymanipulating the nucleic acid template, peptide, polypeptide, andprotein mutations such as substitutions, deletions, truncations,additions, duplications, or fusions of more than one peptide,polypeptide, or protein can be performed. These fusions of more than onepeptide, polypeptide, or protein can be joined in the same moleculeadjacently, or with extra amino acids in between, e.g. linkers, repeats,epitopes, or tags, or any other sequence that is, is about, is at leak,is at least about, is not more than, or is not more than about, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or300 bases long, or any length in a range defined by any two of theaforementioned lengths. The term “downstream” on a polypeptide as usedherein has its plain and ordinary meaning as understood in light of thespecification and refers to a sequence being after the C-terminus of aprevious sequence. The term “upstream” on a polypeptide as used hereinhas its plain and ordinary meaning as understood in light of thespecification and refers to a sequence being before the N-terminus of asubsequent sequence.

The term “purity” of any given substance, compound, or material as usedherein has its plain and ordinary meaning as understood in light of thespecification and refers to the actual abundance of the substance,compound, or material relative to the expected abundance. For example,the substance, compound, or material may be at least 80, 85, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals inbetween. Purity may be affected by unwanted impurities, including butnot limited to nucleic acids, DNA, RNA, nucleotides, proteins,polypeptides, peptides, amino acids, lipids, cell membrane, cell debris,small molecules, degradation products, solvent, carrier, vehicle, orcontaminants, or any combination thereof. In some embodiments, thesubstance, compound, or material is substantially free of host cellproteins, host cell nucleic acids, plasmid DNA, contaminating viruses,proteasomes, host cell culture components, process related components,mycoplasma, pyrogens, bacterial endotoxins, and adventitious agents.Purity can be measured using technologies including but not limited toelectrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR,chromatography, liquid chromatography, gas chromatography, thin layerchromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy,UV-visible spectrometry, infrared spectrometry, mass spectrometry,nuclear magnetic resonance, gravimetry, or titration, or any combinationthereof.

The term “yield” of any given substance, compound, or material as usedherein has its plain and ordinary meaning as understood in light of thespecification and refers to the actual overall amount of the substance,compound, or material relative to the expected overall amount. Forexample, the yield of the substance, compound, or material is, is about,is at least, is at least about, is not more than, or is not more thanabout, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of theexpected overall amount, including all decimals in between. Yield may beaffected by the efficiency of a reaction or process, unwanted sidereactions, degradation, quality of the input substances, compounds, ormaterials, or loss of the desired substance, compound, or materialduring any step of the production.

The term “% w/w” or “% wt/wt” as used herein has its plain and ordinarymeaning as understood in light of the specification and refers to apercentage expressed in terms of the weight of the ingredient or agentover the total weight of the composition multiplied by 100. The term “%v/v” or “% vol/vol” as used herein has its plain and ordinary meaning asunderstood in the light of the specification and refers to a percentageexpressed in terms of the liquid volume of the compound, substance,ingredient, or agent over the total liquid volume of the compositionmultiplied by 100.

Some embodiments described herein relate to pharmaceutical compositionsthat comprise, consist essentially of, or consist of an effective amountof a cell composition described herein and a pharmaceutically acceptablecarrier, excipient, or combination thereof. A pharmaceutical compositiondescribed herein is suitable for human and/or veterinary applications.

As used herein, “pharmaceutically acceptable” has its plain and ordinarymeaning as understood in light of the specification and refers tocarriers, excipients, and/or stabilizers that are nontoxic to the cellor mammal being exposed thereto at the dosages and concentrationsemployed or that have an acceptable level of toxicity. A“pharmaceutically acceptable” “diluent,” “excipient,” and/or “carrier”as used herein have their plain and ordinary meaning as understood inlight of the specification and are intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with administration to humans, cats, dogs, or othervertebrate hosts. Typically, a pharmaceutically acceptable diluent,excipient, and/or carrier is a diluent, excipient, and/or carrierapproved by a regulatory agency of a Federal, a state government, orother regulatory agency, or listed in the U.S. Pharmacopeia or othergenerally recognized pharmacopeia for use in animals, including humansas well as non-human mammals, such as cats and dogs. The term diluent,excipient, and/or “carrier” can refer to a diluent, adjuvant, excipient,or vehicle with which the pharmaceutical composition is administered.Such pharmaceutical diluent, excipient, and/or carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin. Water, saline solutions and aqueousdextrose and glycerol solutions can be employed as liquid diluents,excipients, and/or carriers, particularly for injectable solutions.Suitable pharmaceutical diluents and/or excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike, A non-limiting example of a physiologically acceptable carrier isan aqueous pH buffered solution. The physiologically acceptable carriermay also comprise one or more of the following: antioxidants, such asascorbic acid, low molecular weight (less than about 10 residues)polypeptides, proteins, such as serum albumin, gelatin, immunoglobulins,hydrophilic polymers such as polyvinylpyrrolidone, amino acids,carbohydrates such as glucose, mannose, or dextrins, chelating agentssuch as EDTA, sugar alcohols such as mannitol or sorbitol, salt-formingcounterions such as sodium, and nonionic surfactants such as TWEEN®,polyethylene glycol (PEG), and PLURONTCS®. The composition, if desired,can also contain minor amounts of wetting, bulking, emulsifying agents,or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, sustained release formulations and thelike. The formulation should suit the mode of administration.

Cryoprotectants are cell composition additives to improve efficiency andyield of low temperature cryopreservation by preventing formation oflarge ice crystals. Cryoprotectants include but are not limited to DMSO,ethylene glycol, glycerol, propylene glycol, trehalose, formamide,methyl-formamide, dimethyl-formamide, glycerol 3-phosphate, proline,sorbitol, diethyl glycol, sucrose, triethylene glycol, polyvinylalcohol, polyethylene glycol, or hydroxyethyl starch. Cryoprotectantscan be used as part of a cryopreservation medium, which include othercomponents such as nutrients (e.g. albumin, serum, bovine serum, fetalcalf serum [FCS]) to enhance post-thawing survivability of the cells. Inthese cryopreservation media, at least one cryoprotectant may be foundat a concentration that is, is about, is at least, is at least about, isnot more than, or is not more than about, 0.01%, 0.05%, 0.1%, 0.5%, 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 30%, 40%, 50%, 60%, 70?, 80%, or 90%, or any percentagewithin a range defined by any two of the aforementioned numbers.

Additional excipients with desirable properties include but are notlimited to preservatives, adjuvants, stabilizers, solvents, buffers,diluents, solubilizing agents, detergents, surfactants, chelatingagents, antioxidants, alcohols, ketones, aldehydes,ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodiumchloride, sodium bicarbonate, sodium phosphate, sodium borate, sodiumcitrate, potassium chloride, potassium phosphate, magnesium sulfatesugars, dextrose, fructose, mannose, lactose, galactose, sucrose,sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80,sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate,octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin,esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combinationthereof. Some excipients may be in residual amounts or contaminants fromthe process of manufacturing, including but not limited to serum,albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde,glutaraldehyde, β-propiolactone, gelatin, cell debris, nucleic acids,peptides, amino acids, or growth medium components or any combinationthereof. The amount of the excipient may be found in composition at apercentage that is, is about, is at least, is at least about, is notmore than, or is not more than about, 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w/w or any percentage byweight in a range defined by any two of the aforementioned numbers.

The term “pharmaceutically acceptable salts” has its plain and ordinarymeaning as understood in light of the specification and includesrelatively non-toxic, inorganic and organic acid, or base addition saltsof compositions or excipients, including without limitation, analgesicagents, therapeutic agents, other materials, and the like. Examples ofpharmaceutically acceptable salts include those derived from mineralacids, such as hydrochloric acid and sulfuric acid, and those derivedfrom organic acids, such as ethanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid, and the like. Examples of suitable inorganicbases for the formation of salts include the hydroxides, carbonates, andbicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium,aluminum, zinc, and the like. Salts may also be formed with suitableorganic bases, including those that are non-toxic and strong enough toform such salts. For example, the class of such organic bases mayinclude but are not limited to mono-, di-, and trialkylamines, includingmethylamine, dimethylamine, and triethylamine; mono-, di-, ortrihydroxyalkylamines including mono-, di-, and triethanolamine; aminoacids, including glycine, arginine and lysine; guanidine;N-methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine;morpholine; ethylenediamine; N-benzylphenethylamine; trihydroxymethylaminoethane.

Proper formulation is dependent upon the route of administration chosen.Techniques for formulation and administration of the compounds describedherein are known to those skilled in the art. Multiple techniques ofadministering a compound exist in the art including, but not limited to,enteral, oral, rectal, topical, sublingual, buccal, intraaural,epidural, epicutaneous, aerosol, parenteral delivery, includingintramuscular, subcutaneous, intra-arterial, intravenous, intraportal,intra-articular, intradermal, peritoneal, intramedullary injections,intrathecal, direct intraventricular, intraperitoneal, intranasal orintraocular injections. Pharmaceutical compositions will generally betailored to the specific intended route of administration.

As used herein, a “carrier” has its plain and ordinary meaning asunderstood in light of the specification and refers to a compound,particle, solid, semi-solid, liquid, or diluent that facilitates thepassage, delivery and/or incorporation of a compound to cells, tissuesand/or bodily organs.

As used herein, a “diluent” has its plain and ordinary meaning asunderstood in light of the specification and refers to an ingredient ina pharmaceutical composition that lacks pharmacological activity but maybe pharmaceutically necessary or desirable. For example, a diluent maybe used to increase the bulk of a potent drug whose mass is too smallfor manufacture and/or administration. It may also be a liquid for thedissolution of a drug to be administered by injection, ingestion orinhalation. A common form of diluent in the art is a buffered aqueoussolution such as, without limitation, phosphate buffered saline thatmimics the composition of human blood.

Stem Cells

The term “totipotent stem cells” (also known as omnipotent stem cells)as used herein has its plain and ordinary meaning as understood in lightof the specification and are stem cells that can differentiate intoembryonic and extra-embryonic cell types. Such cells can construct acomplete, viable organism. These cells are produced from the fusion ofan egg and sperm cell. Cells produced by the first few divisions of thefertilized egg are also totipotent.

The term “embryonic stem cells (ESCs),” also commonly abbreviated as EScells, as used herein has its plain and ordinary meaning as understoodin light of the specification and refers to cells that are pluripotentand derived from the inner cell mass of the blastocyst, an early-stageembryo. For purpose of the present disclosure, the term “ESCs” is usedbroadly sometimes to encompass the embryonic germ cells as well.

The term “pluripotent stem cells (PSCs)” as used herein has its plainand ordinary meaning as understood in light of the specification andencompasses any cells that can differentiate into nearly all cell typesof the body, i.e., cells derived from any of the three germ layers(germinal epithelium), including endoderm (interior stomach lining,gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood,urogenital), and ectoderm (epidermal tissues and nervous system). PSCscan be the descendants of inner cell mass cells of the preimplantationblastocyst or obtained through induction of a non-pluripotent cell, suchas an adult somatic cell, by forcing the expression of certain genes.Pluripotent stem cells can be derived from any suitable source. Examplesof sources of pluripotent stem cells include mammalian sources,including human, rodent, porcine, and bovine.

The term “induced pluripotent stem cells (iPSCs),” also commonlyabbreviated as iPS cells, as used herein has its plain and ordinarymeaning as understood in light of the specification and refers to a typeof pluripotent stem cells artificially derived from a normallynon-pluripotent cell, such as an adult somatic cell, by inducing a“forced” expression of certain genes. hiPSC refers to human iPSCs. Insome methods known in the art, iPSCs may be derived by transfection ofcertain stem cell-associated genes into non-pluripotent cells, such asadult fibroblasts. Transfection may be achieved through viraltransduction using viruses such as retroviruses or lentiviruses.Transfected genes may include the master transcriptional regulatorsOct-3/4 (POU5F1) and Sox2, although other genes may enhance theefficiency of induction. After 3-4 weeks, small numbers of transfectedcells begin to become morphologically and biochemically similar topluripotent stem cells, and are typically isolated through morphologicalselection, doubling time, or through a reporter gene and antibioticselection. As used herein, iPSCs include first generation iPSCs, secondgeneration iPSCs in mice, and human induced pluripotent stem cells. Insome methods, a retroviral system is used to transform human fibroblastsinto pluripotent stem cells using four pivotal genes: Oct3/4, Sox2,K1f4, and c-Myc. In other methods, a lentiviral system is used totransform somatic cells with OCT4, SOX2, NANOG, and LIN28. Genes whoseexpression are induced in iPSCs include but are not limited to Oct-3/4(POU5F1); certain members of the Sox gene family (e.g., Sox1, Sox2,Sox3, and Sox15); certain members of the K1f family (e.g., K1f1, K1f2,K1f4, and K1f5), certain members of the Myc family (e.g., C-myc, L-myc,and N-myc), Nanog, LIN28, Tert, Fbx15, ERas, ECAT15-1, ECAT15-2, Tcl1,β-Catenin, ECAT1, Esg1; Dnmt3L, ECAT8, Gdf3, Fth117, Sa114, Rex1; UTF1,Stella, Stat3, Grb2, Prdm14, Nr5a1, Nr5a2, or E-cadherin, or anycombination thereof.

The term “precursor cell” as used herein has its plain and ordinarymeaning as understood in light of the specification and encompasses anycells that can be used in methods described herein, through which one ormore precursor cells acquire the ability to renew itself ordifferentiate into one or more specialized cell types. In someembodiments, a precursor cell is pluripotent or has the capacity tobecoming pluripotent. In some embodiments, the precursor cells aresubjected to the treatment of external factors (e.g., growth factors) toacquire pluripotency. In some embodiments, a precursor cell can be atotipotent (or omnipotent) stem cell; a pluripotent stem cell (inducedor non-induced); a multipotent stem cell; an oligopotent stem cells anda unipotent stem cell. In some embodiments, a precursor cell can be froman embryo, an infant, a child, or an adult. In some embodiments, aprecursor cell can be a somatic cell subject to treatment such thatpluripotency is conferred via genetic manipulation or protein/peptidetreatment. Precursor cells include embryonic stem cells (ESC), embryoniccarcinoma cells (ECs), and epiblast stem cells (EpiSC).

In some embodiments, one step is to obtain stem cells that arepluripotent or can be induced to become pluripotent. In someembodiments, pluripotent stem cells are derived from embryonic stemcells, which are in turn derived from totipotent cells of the earlymammalian embryo and are capable of unlimited, undifferentiatedproliferation in vitro. Embryonic stem cells are pluripotent stem cellsderived from the inner cell mass of the blastocyst, an early-stageembryo. Methods for deriving embryonic stem cells from blastocytes arewell known in the art. Human embryonic stem cells H9 (H9-hESCs) are usedin the exemplary embodiments described in the present application, butit would be understood by one of skill in the art that the methods andsystems described herein are applicable to any stem cells.

Additional stem cells that can be used in embodiments in accordance withthe present dislcosure include but are riot limited to those provided byor described in the database hosted by the National Stem Cell Bank(NSCB), Human Embryonic Stem Cell Research Center at the University ofCalifornia, San Francisco (UCSF); WISC cell Bank at the Wi Cell ResearchInstitute; the University of Wisconsin Stem Cell and RegenerativeMedicine Center (UW-SCRMC); Novocell, Inc. (San Diego, Calif.);Cellartis AB (Goteborg, Sweden); ES Cell International Pte Ltd(Singapore); Technion at the Israel Institute of Technology (Haifa,Israel); and the Stem Cell Database hosted by Princeton University andthe University of Pennsylvania. Exemplary embryonic stem cells that canbe used in embodiments in accordance with the present disclosure includebut are not limited to SA01 (SA001); SA02 (SA002); ES01 (HES-1); ES02(HES-2); ES03 (HES-3); ES04 HES-4); ES05 (HES-5); ES06 HES-6); BG01(BGN-01); BG02 (BGN-02); BG-03 (BGN-03); TE03 (13); TE04 (14); TE06(16); UCO1 (HSF1); UC06 (HSF6); WA01 (HI); WA07 (H7); WA09 (H9); WA13(H13); WA14 (H14). Exemplary human pluripotent cell lines include butare not limited to TkDA3-4, 1231A3, 317-D6, 317-A4, CDH1, 5-T-3, 3-34-1,NAFLD27, NAFLD77, NAFLD150, WD90, WD91, WD92, L20012, C213, 1383D6, FF,or 317-12 cells.

In developmental biology, cellular differentiation is the process bywhich a less specialized cell becomes a more specialized cell type. Asused herein, the term “directed differentiation” describes a processthrough which a less specialized cell becomes a particular specializedtarget cell type. The particularity of the specialized target cell typecan be determined by any applicable methods that can be used to defineor alter the destiny of the initial cell. Exemplary methods include butare not limited to genetic manipulation, chemical treatment, proteintreatment, and nucleic acid treatment.

In some embodiments, an adenovirus can be used to transport therequisite four genes, resulting in iPSCs substantially identical toembryonic stem cells. Since the adenovirus does not combine any of itsown genes with the targeted host, the danger of creating tumors iseliminated. In some embodiments, non-viral based technologies areemployed to generate iPSCs. In some embodiments, reprogramming can beaccomplished via plasmid without any virus transfection system at all,although at very low efficiencies. In other embodiments, direct deliveryof proteins is used to generate iPSCs, thus eliminating the need forviruses or genetic modification. In some embodiment, generation of mouseiPSCs is possible using a similar methodology: a repeated treatment ofthe cells with certain proteins channeled into the cells viapoly-arginine anchors was sufficient to induce pluripotency. In someembodiments, the expression of pluripotency induction genes can also beincreased by treating somatic cells with FGF2 under low oxygenconditions.

The term “feeder cell” as used herein has its plain and ordinary meaningas understood in light of the specification and refers to cells thatsupport the growth of pluripotent stem cells, such as by secretinggrowth factors into the medium or displaying on the cell surface. Feedercells are generally adherent cells and may be growth arrested. Forexample, feeder cells are growth-arrested by irradiation (e.g. gammarays), mitomycin-C treatment, electric pulses, or mild chemical fixation(e.g. with formaldehyde or glutaraldehyde). However, feeder cells do notnecessarily have to be growth arrested. Feeder cells may serve purposessuch as secreting growth factors, displaying growth factors on the cellsurface, detoxifying the culture medium, or synthesizing extracellularmatrix proteins. In some embodiments, the feeder cells are allogeneic orxenogeneic to the supported target stem cell, which may haveimplications in downstream applications. In some embodiments, the feedercells are mouse cells. In some embodiments, the feeder cells are humancells. In some embodiments, the feeder cells are mouse fibroblasts,mouse embryonic fibroblasts, mouse STO cells, mouse 3T3 cells, mouse SNL76/7 cells, human fibroblasts, human foreskin fibroblasts, human dermalfibroblasts, human adipose mesenchymal cells, human bone marrowmesenchymal cells, human amniotic mesenchymal cells, human amnioticepithelial cells, human umbilical cord mesenchymal cells, human fetalmuscle cells, human fetal fibroblasts, or human adult fallopian tubeepithelial cells. In some embodiments, conditioned medium prepared fromfeeder cells is used in lieu of feeder cell co-culture or in combinationwith feeder cell co-culture. In some embodiments, feeder cells are notused during the proliferation of the target stem cells.

Blood cell production derives from a single type of cell, thehematopoietic stem cell, which through proliferation anddifferentiation, gives rise to the entire hematopoietic system. Thehematopoietic stem cells are believed to be capable of self-renewal,expanding their own population of stem cells, and they are pluripotent(capable of differentiating into any cell in the hematopoietic system).From this rare cell population, the entire mature hematopoietic system,comprising lymphocytes (B and T cells of the immune system) and myeloidcells (erythrocytes, megakaryocytes, granulocytes and macrophages) isformed. The lymphoid lineage, comprising B cells and T cells, providesfor the production of antibodies, regulation of the cellular immunesystem, detection of foreign agents in the blood, detection of cellsforeign to the host, and the like. The myeloid lineage, which includesmonocytes, granulocytes, megakaryocytes as well as other cells, monitorsfor the presence of foreign bodies, provides protection againstneoplastic cells, scavenges foreign materials, produces platelets, andthe like. The erythroid lineage provides red blood cells, which act asoxygen carriers.

The liver is a vital organ that provides many essential metabolicfunctions for life such as the detoxification of exogenous compounds andcoagulation as well as producing lipids, proteins, ammonium, and bile.Primary hepatocytes are a highly polarized metabolic cell type, and forma bile canaliculi structure with micro villi-lined channels, separatingperipheral circulation from the bile acid secretion pathway. In vitroreconstitution of a patient's liver may provide applications includingregenerative therapy, drug discovery and drug toxicity studies. Existingmethodology using primary liver cells exhibit extremely poorfunctionality, largely due to a lack of essential anatomical structures,which limits their practical use for the pharmaceutical industry. Theformation of liver organoids, which comprise a luminal structure withinternalized microvilli and mesenchymal cells, as well as exhibit livercell types such as hepatocytes, stellate cells, Kupffer cells, and liverendothelial cells, and methods of making and use thereof have previouslybeen described in PCT Publications WO2018/085615, WO2018/085622,WO2018/085623, and WO2018/226267, each of which is hereby expresslyincorporated by reference in its entirety.

In some embodiments, ESCs, germ cells, or iPSCs are cultured in growthmedia that supports the growth of stem cells. In some embodiments, theESCs, germ cells, or iPSCs are cultured in stem cell growth media. Insome embodiments, the stem cell growth media is RPM1 1640, DMEM,DMEM/F12, Advanced DMEM, hepatocyte culture medium (HCM), StemFit, mTeSR1, or mTeSR Plus media. In some embodiments, the stem cell growth mediacomprises fetal bovine serum (FBS). In some embodiments, the stem cellgrowth media comprises FBS at a concentration that is, is about, is atleast, is at least about, is not more than, or is not more than about,0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, or 20%, or any percentage within a range defined by any two of theaforementioned concentrations, for example 0% to 20%, 0.2% to 10%, 2% to5%, 0% to 5%, or 2% to 20%. In some embodiments, the stem cell growthmedia does not contain xenogeneic components. In some embodiments, thegrowth media comprises one or more small molecule compounds, activators,inhibitors, or growth factors. In some embodiments, the stem cells aregrown on a feeder cell substrate. In some embodiments, the stem cellsare not grown on a feeder cell substrate. In some embodiments, the stemcells are grown on plates coated with laminin. In some embodiments, thestem cells are grown supplemented with FGF2 or a ROCK inhibitor (e.g.Y-27632), or both. In some embodiments, the FGF2 is at a concentrationthat is, is about, is at least, is at least about, is not more than, oris not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,300, 400, or 500 ng/mL, or any concentration within a range defined byany two of the aforementioned concentrations, for example, 1 to 500ng/mL, 10 to 200 ng/mL, 100 to 150 ng/mL, 1 to 100 ng/mL, or 100 to 500ng/mL. In some embodiments, the ROCK inhibitor is at a concentrationthat is, is about, is at least, is at least about, is not more than, oris not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nM, orany concentration within a range defined by any two of theaforementioned concentrations, for example, 1 to 30 nM, 5 to 25 nM, 10to 20 nM, 1 to 15 nM, or 10 to 30 nM. In some embodiments, the stemcells are grown on plates coated with Laminin.

Methods of Definitive Endoderm Differentiation

Any methods for producing definitive endoderm (DE) from pluripotentcells (e.g., iPSCs or ESCs) are applicable to the methods describedherein. Exemplary methods are disclosed in, for example, U.S. Pat. No.9,719,068. In some embodiments, iPSCs are used to produce definitiveendoderm.

In some embodiments, one or more growth factors are used in thedifferentiation process from pluripotent stem cells to DE cells. In someembodiments, the one or more growth factors used in the differentiationprocess include growth factors from the TGF-beta superfamily. In someembodiments, the one or more growth factors comprise the Nodal/Activinand/or the BMP subgroups of the TGF-beta superfamily of growth factors.In some embodiments, the one or more growth factors are selected fromthe group consisting of Nodal, Activin A, Activin B, BMP4, or anycombination thereof. In some embodiments, the PSCs are contacted withthe one or more growth factors for a number of days that is, is about,is at least, is at least about, is not more than, or is not more thanabout, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, or 240hours, or any number of hours within a range defined by any two of theaforementioned number of days, for example, 1 to 240 hours, 20 to 120hours. 30 to 50 hours, 1 to 100 hours, or 50 to 240 hours. In someembodiments, the PSCs are contacted with the one or more growth factorsat a concentration that is, is about, is at least, is at least about, isnot more than, or is not more than about, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 ng/mL, orany concentration within a range defined by any two of theaforementioned concentrations, for example, 10 to 1000 ng/mL, 50 to 800ng/mL, 100 to 500 ng/mL, 10 to 200 ng/mL or 100 to 1000 ng/mL. In someembodiments, the concentration of the one or more growth factors ismaintained at a constant level through the period of contacting. In someembodiments, the concentration of the one or more growth factors isvaried during the period of contacting. In some embodiments, the one ormore growth factors is dissolved into the growth media. In someembodiments, populations of cells enriched in definitive endoderm cellsare used. In some embodiments, the definitive endoderm cells areisolated or substantially purified. In some embodiments, the isolated orsubstantially purified definitive endoderm cells express one or more(e.g. at least 1, 3) of SOX17, FOXA2, or CXRC4 markers to a greaterextent than one or more (e.g. at least 1, 3, 5) of OCT4, AFP, TM, SPARC,or SOX7 markers.

In some embodiments, the definitive endoderm cells are contacted withone or more modulators of a signaling pathway described herein. In someembodiments, the definitive endoderm cells are treated with the one ormore modulators of a signaling pathway for a number of days that is, isabout, is at least, is at least about, is not more than, or is not morethan about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, hours, or 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, orany number of hours or days within a range defined by any two of theaforementioned number of days or hours, for example, 1 hour to 20 days,20 hours to 10 days, 1 hour to 48 hours, 1 day to 20 days, 1 hour to 5days, or 24 hours to 20 days. In some embodiments, the concentration ofthe one or more modulators of a signaling pathway is maintained at aconstant level through the period of contacting. In some embodiments,the concentration of the one or more modulators of a signaling pathwayis varied during the period of contacting.

In some embodiments, to differentiate the definitive endoderm intoforegut spheroids, the definitive endoderm cells are contacted with oneor more modulators of an FGF pathway and a Wnt pathway. In someembodiments, cellular constituents associated with the Wnt and/or FGFsignaling pathways, for example, natural inhibitors, antagonists,activators, or agonists of the pathways can be used to result ininhibition or activation of the Wnt and/or FGF signaling pathways. Insome embodiments, siRNA and/or shRNA targeting cellular constituentsassociated with the Wnt and/or FGF signaling pathways are used toinhibit or activate these pathways.

Fibroblast growth factors (FGFs) are a family of growth factors involvedin angiogenesis, wound healing, and embryonic development. The FGFs areheparin-binding proteins and interactions with cell-surface associatedheparan sulfate proteoglycans have been shown to be essential for FGFsignal transduction. FGFs are key players in the processes ofproliferation and differentiation of wide variety of cells and tissues.In humans, 22 members of the FGF family have been identified, all ofwhich are structurally related signaling molecules. Members FGF1 throughFGF10 all bind fibroblast growth factor receptors (FGFRs). MU is alsoknown as acidic, and FGF2 is also known as basic fibroblast growthfactor (bFGF). Members FGF11, FGF12, FGF13, and FGF14, also known as FGFhomologous factors 1-4 (FHF1-FHF4), have been shown to have distinctfunctional differences compared to the FGFs. Although these factorspossess remarkably similar sequence homology, they do not bind FGFRs andare involved in intracellular processes unrelated to the FGFs. Thisgroup is also known as “iFGE” Members FGF15 through FGF23 are newer andnot as well characterized. FGF15 is the mouse ortholog of human FGF19(hence there is no human FGF15). Human FGF20 was identified based on itshomology to Xenopus FGF-20 (XFGF-20). In contrast to the local activityof the other FGFs, FGF15/FGF19, FGF21 and FGF23 have more systemiceffects. In some embodiments, the FGF used is one or more (e.g. at least1, 3, 5) of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8,FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15 (FGF19, FGF15/FGF19),FGF16, FGF17, FGF18, FGF20, FGF21, FGF22, FGF23. In some embodiments,the FGF used is FGF4. In some embodiments, the definitive endoderm iscontacted with an FGF at a concentration that is, is about, is at least,is at least about, is not more than, or is not more than about, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000ng/mL, or any concentration within a range defined by any two of theaforementioned concentrations, for example, 10 to 2000 ng/mL, 50 to 1500ng/mL, 500 to 100 ng/mL, 10 to 1000 ng/mL or 500 to 2000 ng/mL.

In some embodiments, to differentiate the definitive endoderm intoforegut spheroids, the definitive endoderm is contacted with a Wntprotein or activator. In some embodiments, the definitive endoderm iscontacted with a glycogen synthase kinase 3 (GSK3) inhibitor. GSK3inhibitor act to activate Wnt pathways. In some embodiments, thedefinitive endoderm is contacted with the GSK3 inhibitor Chiron(CHIR99021). In some embodiments, the definitive endoderm is contactedwith CHIR99021 at a concentration that is, is about, is at least, is atleast about, is not more than, or is not more than about, 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM ofCHIR99021 or any concentration within a range defined by any two of theaforementioned concentrations, for example, 0.1 to 10 μM, 0.4 to 6 μM, 1to 5 μμM, 0.1 to 1 μM, or 0.5 to 10 μM of CHIR99021.

In some embodiments, the foregut spheroids comprise a mesoderm. In someembodiments, the foregut spheroids comprise mesoderm cells. In someembodiments, the foregut spheroids comprise mesodermal progenitor cells.In some embodiments, the foregut spheroids comprise mesenchyme. In someembodiments, the foregut spheroids comprise mesenchymal cells. In someembodiments, the foregut spheroids comprise mesenchymal stem cells. Insome embodiments, the foregut spheroids comprise mesenchymal progenitorcells. In some embodiments, one or more (e.g. at least 1, 2, 3, 4, 5) ofthe mesoderm, mesoderm cells, mesodermal progenitor cells, mesenchyme,mesenchymal cells, mesenchymal stem cells, or mesenchymal progenitorcells, or any combination thereof, differentiate to a hemogenicendothelium population. In some embodiments, the mesenchymedifferentiates into a hemogenic endothelium population. In someembodiments, the hemogenic endothelium population is a CD34⁺ hemogenicendothelium population. In some embodiments, the hemogenic endotheliumpopulation is the hemogenic endothelium population of any one of theliver organoids described herein.

Methods of Foregut Spheroid Differentiation

In some embodiments, the foregut spheroids are differentiated into liverorganoids. In some embodiments, the foregut spheroids are differentiatedinto liver organoids by contacting the foregut spheroids with retinoicacid (RA). In some embodiments, the liver organoids produced fromcontacting the foregut spheroids resemble mature liver tissue. In someembodiments, the foregut spheroids are differentiated into liverorganoids that more closely resemble fetal liver tissue. In someembodiments, the foregut spheroids are differentiated into liverorganoids that more closely resemble fetal liver tissue by notcontacting the foregut spheroids with RA.

In some embodiments, fetal liver organoids are produced by a Matrigeldrop method. In some embodiments, foregut spheroids are embedded in 100%Matrigel and incubated at 37° C. to solidify the Matrigel. In someembodiments, the foregut spheroids embedded in Matrigel are grown inmedia that does not comprise retinoic acid (e.g. DMEM/F12), for a numberof days that is, is about, is at least, is at least about, is not morethan, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30days, or any number of days within a range defined by any two of theaforementioned number of days, for example, 1 to 30 days, 10 to 20 days,12 to 18 days, 1 to 20 days, or 10 to 30 days. In some embodiments, theforegut spheroids are grown in media that does not comprise one or moreof dexamethasone, Oncostatin M, or hepatocyte growth factor, or anycombination thereof.

In some embodiments, the foregut spheroids or liver organoids, or both,are contacted with a cytokine. In some embodiments, the cytokine istransferring, stem cell factor (SCF), interleukin 3 (IL-3), interleukin6 (IL-6), interleukin 34 (IL-34), erythropoietin (EPO), granulocytecolony-stimulating factor (G-CSF), granulocyte-macrophagecolony-stimulating factor (GM-CSF), or any combination thereof In someembodiments, the foregut spheroids or liver organoids, or both, aredissociated into single cells before contacting with the cytokine. Insome embodiments, the cytokine is at a concentration that is, is about,is at least, is at least about, is not more than, or is not more thanabout, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000 ng/mL, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000 μg/mL, or any concentration within a rangedefined by any two of the aforementioned concentrations, for example, 1ng/mL to 1000 ng/mL, 50 ng/mL to 100 μg/mL, 500 ng/mL to 10 μg/mL, 1ng/mL to 1000 ng/mL, 1 μg/mL to 1000 μg/mL, 1 ng/mL, to 50 μg/mL, or 100ng/mL to 1000 μg/mL. In some embodiments, the foregut spheroids or liverorganoids, or both, are contacted with the cytokine for a number of daysthat is, is about, is at least, is at least about, is not more than, oris not more than about, 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50days, or any number of days within a range defined by any two of theaforementioned number of days, for example, 1 to 50 days, 5 to 40 days,10 to 30 days, 1 to 30 days, or 20 to 50 days.

In some embodiments, the foregut spheroids or liver organoids, or both,are contacted with a Notch activator or ligand. Notch signaling isimportant in establishing definitive hematopoiesis in hemogenicendothelium, and a Notch activator or ligand activates the Notchsignaling pathway. In some embodiments, a Notch ligand is a Notchactivator. In some embodiments, contact with the Notch activator orligand increases the production of CD45⁺ hematopoietic cells in theforegut spheroids, or liver organoids, or both. In some embodiments,contact with the Notch activator or ligand increases the number oferythrocytes produced by the foregut spheroids, or liver organoids, orboth. In some embodiments, contact with the Notch activator or ligandincreases expression of IL-6 in the foregut spheroids, or liverorganoids, or both. In some embodiments, contact with the Notchactivator or ligand increases expression of HES1 in the foregutspheroids, or liver organoids, or both. In some embodiments, the Notchactivator or ligand comprises one or more (e.g. at least 1, 2, 3, 4, 5)of DIL1, DLL3, DLL4, JAG1, or JAG2, or any combination thereof. In someembodiments, the Notch activator or ligand comprises a cell expressingone or more (e.g. at least 1, 2, 3, 4, 5) of DLL1, DLL3, DLL4, JAG1, orJAG2, or any combination thereof. In some embodiments, the foregutspheroid comprises a cell that expresses one or more (e.g. at least 1,2, 3, 4, 5) Notch activators or ligands selected from the groupconsisting of DLL1, DLL3, DLL4, JAG1, or JAG2, or any combinationthereof In some embodiments, the foregut spheroid expresses one or more(e.g. at least 1, 2, 3, 4, 5) Notch activators selected from the groupconsisting of DLL1, DLL3, DLL4, JAG1, or JAG2, or any combinationthereof. In some embodiments, the Notch activator or ligand is DLL4. Insome embodiments, the foregut spheroids or liver organoids, or both, aredissociated into single cells before contacting with the Notch activatoror ligand. In some embodiments, the Notch activator or ligand is at aconcentration that is, is about, is at least, is at least about, is notmore than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000 ng/mL, or 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 μg/mL, or anyconcentration within a range defined by any two of the aforementionedconcentrations, for example, 1 ng/mL to 1000 μg/mL, 50 ng/mL to 100mg/mL, 500 ng/mL to 10 μg/mL, 1 ng/mL to 1000 ng/mL, 1 μg/mL to 1000μg/mL, 1 ng/mL to 50 μg/mL, or 100 ng/mL to 1000 μg/mL. In someembodiments, the foregut spheroids or liver organoids, or both, arecontacted with the Notch activator or ligand for a number of days thatis, is about, is at least, is at least about, is not more than, or isnot more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50days, or any number of days within a range defined by any two of theaforementioned number of days, for example, 1 to 50 days, 5 to 40 days,10 to 30 days, 1 to 30 days, or 20 to 50 days.

In some embodiments, albumin secretion of the foregut spheroids or liverorganoids, or both, is quantified. In some embodiments, albuminsecretion is quantified by ELISA, In some embodiments, the foregutspheroids or liver organoids, or both, are examined byimmunohistochemistry. In some embodiments, the foregut spheroids, orliver organoids, or both, are examined for albumin, CD34, or CD45expression, or any combination thereof. In some embodiments, the foregutspheroids, or liver organoids, or both, are examined by flow cytometry.In some embodiments, the foregut spheroids, or liver organoids, or bothare examined for CD45, CD34, CD11b, CD19, CD27, CD43, or CD45expression, or any combination thereof. In some embodiments, the foregutspheroids, or liver organoids, or both are examined with a hematopoieticcolony forming cell assay. In some embodiments, the foregut spheroids,or liver organoids, or both, produce erythroid colonies, myeloidcolonies, or lymphoid colonies, or any combination thereof. In someembodiments, the foregut spheroids, or liver organoids, or both, areexamined by RT-qPCR, In some embodiments, RT-qPCR is performed using thesequences of SEQ ID NO:1-12. In some embodiments, the foregut spheroids,or liver organoids, or both, are examined by RT-qPCR to quantifyexpression of AFP, ALB, CD34, HBG1, HES1, or HES5, or any combinationthereof. In some embodiments, the foregut spheroids, or liver organoids,or both, are examined by single cell RNA sequencing.

In some embodiments, methods of producing a liver organoid that produceshematopoietic stem cells (HSCs) are provided. In some embodiments, themethods comprise culturing induced pluripotent stem cells underconditions sufficient to differentiate the induced pluripotent stemcells into definitive endoderm, culturing the definitive endoderm underconditions sufficient to differentiate the definitive endoderm into aforegut spheroid, and culturing the foregut spheroid under conditionssufficient to differentiate the foregut spheroid into a liver organoid.In some embodiments, the methods comprise culturing induced pluripotentstem cells under conditions sufficient to differentiate the inducedpluripotent stem cells into definitive endoderm. In some embodiments,the methods comprise culturing a definitive endoderm under conditionssufficient to differentiate the definitive endoderm into a foregutspheroid. In some embodiments, the methods comprise culturing a foregutspheroid under conditions sufficient to differentiate the foregutspheroid into a liver organoid. In some embodiments, culturing theforegut spheroid under conditions sufficient to differentiate theforegut spheroid into a liver organoid comprises contacting the foregutspheroid with one or more (e.g. at least 1, 2, 3) of an FGF signalingpathway activator, a Wtn signaling pathway activator, or a BMPinhibitor, or any combination thereof. In some embodiments, culturingthe foregut spheroid under conditions sufficient to differentiate theforegut spheroid into a liver organoid comprises contacting the foregutspheroid with an FGF signaling pathway activator or a Wnt signalingpathway activator, or both. In some embodiments, the FGF signalingpathway activator is FGF4, In some embodiments, the Wnt signalingpathway activator is CHIR99021. In some embodiments, the BMP inhibitoris Noggin. In some embodiments, the foregut spheroid comprises amesoderm. In some embodiments, the foregut spheroid comprises mesodermcells. In some embodiments, the foregut spheroid comprises mesodermalprogenitor cells. In some embodiments, the foregut spheroid comprisesmesenchyme. In some embodiments, the foregut spheroid comprisesmesenchymal cells. In some embodiments, the foregut spheroid comprisesmesenchymal stem cells. In some embodiments, the foregut spheroidcomprises mesenchymal progenitor cells. In some embodiments, one or more(e.g. at least 1, 2, 3, 4, 5) of the mesoderm, mesoderm cells,mesodermal progenitor cells, mesenchyme, mesenchymal cells, mesenchymalstem cells, mesenchymal stem cells or mesenchymal progenitor cells, orany combination thereof, differentiate to a hemogenic endotheliumpopulation. In some embodiments, the hemogenic endothelium population isa CD34⁺ hemogenic endothelium population. In some embodiments, the liverorganoid comprises a CD34⁺ hemogenic endothelium population. In someembodiments, the hemogenic endothelium population differentiates intohematopoietic stem cells. In some embodiments, the hemogenic endotheliumpopulation of the liver organoid is differentiated from one or more(e.g. at least 1, 2, 3, 4, 5) of the mesoderm, mesoderm cells,mesodermal progenitor cells, mesenchyme, mesenchymal cells, mesenchymalstem cells, or mesenchymal progenitor cells, or any combination thereof,of the foregut spheroid. In some embodiments, the hemogenic endotheliumpopulation of the liver organoid is differentiated from the mesenchymeof the foregut spheroid. In some embodiments, the hemogenic endotheliumpopulation differentiates into erythrocytes. In some embodiments, thehemogenic endothelium population differentiates into erythrocyteswithout the addition of exogenous hematopoietic cytokines. In someembodiments, the liver organoid comprises one or more (e.g. at least 1,2, 3) of a hepatic population, an erythroid population, or a hemogenicendothelium population, or any combination thereof. In some embodiments,the liver organoid comprises cells that express EpCAM. In someembodiments, the liver organoid comprises CD34⁺ endothelium. In someembodiments, the CD34⁺ endothelium expresses one or more (e.g. at least1, 3, 5) of Notch1, Notch4, HEY1, HEY2, DLL4, or GATA2, or anycombination thereof. In some embodiments, the liver organoid compriseserythroid cells expressing GYPA. In some embodiments, the liver organoidcomprises CD45-expressing hematopoietic cells.

In some embodiments, the induced pluripotent stem cells are cultured fora number of days that is, is about, is at least, is at least about, isnot more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, or 50 days, or any number of days within a range defined byany two of the aforementioned number of days, for example, 1 to 50 days,5 to 40 days, 10 to 30 days, 1 to 30 days, or 20 to 50 days. In someembodiments, the definitive endoderm is cultured for a number of daysthat is, is about, is at least, is at least about, is not more than, oris not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50 days, or any number of days within a range defined by any two of theaforementioned number of days, for example, 1 to 50 days, 5 to 40 days,10 to 30 days, 1 to 30 days, or 20 to 50 days. In some embodiments, theforegut spheroids are cultured for a number of days that is, is about,is at least, is at least about, is not more than, or is not more thanabout, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 days, or anynumber of days within a range defined by any two of the aforementionednumber of days, for example, 1 to 50 days, 5 to 40 days, 10 to 30 days,1 to 30 days, or 20 to 50 days.

In some embodiments, culturing the induced pluripotent stem cellscomprise contacting the induced pluripotent stem cells with one or more(e.g. at least 1 or 2) of Activin A or BNIP4. In some embodiments,culturing the definitive endoderm comprises contacting the definitiveendoderm with one or more (e.g. at least 1 or 2) of CHIR99021 or FGF. Insome embodiments, culturing the foregut spheroid comprises contactingthe foregut spheroid with one or more of BMP4 and FGF2. In someembodiments, culturing the foregut spheroid comprises contacting theforegut spheroid with one or more of BMP4 and FGF2 in an extracellularmatrix. In some embodiments, culturing the foregut spheroid comprisescontacting the foregut spheroid with a Notch activator or ligand. Insome embodiments, the Notch activator or ligand comprises one or more(e.g. at least 1, 3, 5) of DLL1, DLL3, DLL4, JAG1, or JAG2, or anycombination thereof. In some embodiments, the Notch activator or ligandcomprises a cell that expresses one or more (e.g. at least 1, 3, 5) ofDLL1, DLL3, DLL4, JAG1, or JAG2, or any combination thereof. In someembodiments, the foregut spheroid comprises a cell that expresses one ormore (e.g. at least 1, 2, 3, 4, 5) Notch activators or ligands selectedfrom the group consisting of DLL1, DLL3, DLL4, JAG1, or JAG2, or anycombination thereof. In some embodiments, the foregut spheroid expressesone or more (e.g. at least 1, 2, 3, 4, 5) Notch activators or ligandsselected from the group consisting of DLL1, DLL3, DLL4, JAG1, or JAG2,or any combination thereof. In some embodiments, the Notch activator orligand is DLL4. In some embodiments, culturing the foregut spheroidcomprises contacting the foregut spheroid with one or more (e.g. atleast 1, 2, 3) of IL-3, IL-34, or GM-CSF, or any combination thereof. Insome embodiments, culturing the foregut spheroid comprises contactingthe foregut spheroid with stem cell factor (SCF) or thrombopoietin(TPO), or both, In some embodiments, culturing the foregut spheroidfurther comprises contacting the foregut spheroid with one or more (e.g.at least 1, 2, 3, 4, 5) of IL-3, IL-34, EPO, G-CSF, GM-CSF, SCF, or TPO,or any combination thereof. In some embodiments, culturing the foregutspheroid does not comprise contacting the foregut spheroid with retinoicacid (RA). In some embodiments, culturing the foregut spheroid does notcomprise contacting the foregut spheroid with one or more (e.g. at least1, 2, 3 4) of RA, dexamethasone, Oncostatin M, or hepatocyte growthfactor, or any combination thereof.

Described herein are methods of producing a liver organoid that produceshematopoietic stem cells. The methods comprise culturing a foregutspheroid under conditions sufficient to differentiate the foregutspheroid into a liver organoid. In some embodiments, the foregutspheroid comprises a mesenchyme. In some embodiments, the liver organoidcomprises a CD34⁺ hetnogenic endothelium population that differentiateinto hematopoietic stem cells. In some embodiments, the CD34⁺ hemogenicendothelium population originates from the mesenchyme of the foregutspheroid. In some embodiments, the foregut spheroid is obtained byculturing PSCs, iPSCs, or definitive endoderm according to any one ofthe methods described herein. In some embodiments, PSCs or iPSCs arecultured according to any one of the methods described herein for anumber of days that is, is about, is at least, is at least about, is notmore than, or is not more than about, 1, 2, 3, 4, or 5 days to obtaindefinitive endoderm. In some embodiments. PSCs or iPSCs are culturedwith Activin A or BMP4, or both, for a number of days that is, is about,is at least, is at least about, is not more than, or is not more thanabout, 1, 2, 3, 4, or 5 days to obtain definitive endoderm. In someembodiments, definitive endoderm is cultured according to any one of themethods described herein for a number of days that is, is about, is atleast, is at least about, is not more than, or is not more than about,1, 2, 3, 4, or 5 days to obtain foregut spheroids. In some embodiments,definitive endoderm is cultured with FGF4 or CHIR99021, or both, for anumber of days is, is about, is at least, is at least about, is not morethan, or is not more than about, 1, 2, 3, 4, or 5 days to obtain foregutspheroids. In some embodiments, culturing the foregut spheroid comprisescontacting the foregut spheroid with a Notch activator or ligand. Insome embodiments, the Notch activator or ligand is provided at aconcentration that is, is about, is at least, is at least about, is notmore than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, or 50 μg/mL, or any concentration within a rangedefined by any two of the aforementioned concentrations, for example, 1ng/mL to 50 μg/mL, 5 ng/mL to 40 μg/mL, 10 ng/mL to 30 μg/mL, 1 ng/mL to20 ng/mL, or 5 μg/mL to 20 μg/mL. In some embodiments, the foregutspheroid is contacted with the Notch activator or ligand for a number ofdays that is, is about, is at least, is at least about, is not morethan, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 days, or any number of days within arange defined by any two of the aforementioned number of days, forexample, 1 to 20 days, 5 to 15 days, 10 to 14 days, 1 to 15 days, or 5to 20 days.

Described herein are methods of producing a liver organoid that produceshematopoietic stem cells. The methods comprise culturing a foregutspheroid under conditions sufficient to differentiate the foregutspheroid into a liver organoid. In some embodiments, the foregutspheroid comprises a mesenchyme. In some embodiments, the liver organoidcomprises a CD34⁺ hemogenic endothelium population that differentiateinto hematopoietic stem cells. In some embodiments, the CD34⁺ hemogenicendothelium population originates from the mesenchyme of the foregutspheroid. In some embodiments, the foregut spheroid is obtained byculturing PSCs, iPSCs, or definitive endoderm according to any one ofthe methods described herein. In some embodiments, PSCs or iPSCs arecultured according to any one of the methods described herein for anumber of days that is, is about, is at least, is at least about, is notmore than, or is not more than about, 1, 2, 3, 4, or 5 days to obtaindefinitive endoderm. In some embodiments, PSCs or iPSCs are culturedwith Activin A or BMP4, or both, for a number of days that is, is about,is at least, is at least about, is not more than, or is not more thanabout, 1, 2, 3, 4, or 5 days to obtain definitive endoderm. In someembodiments, definitive endoderm is cultured according to any one of themethods described herein for a number of days that is, is about, is atleast, is at least about, is not more than, or is not more than about,1, 2, 3, 4, or 5 days to obtain foregut spheroids. In some embodiments,definitive endoderm is cultured with FGF4 or CHIR99021, or both, for anumber of days is, is about, is at least, is at least about, is not morethan, or is not more than about, 1, 2, 3, 4, or 5 days to obtain foregutspheroids. In some embodiments, culturing the foregut spheroid comprisescontacting the foregut spheroid with one or more (e.g. at least 1, 2, 3,4, 5) of the Notch activators or ligands DLL1, DLL3, DLL4, JAG-1, orJAG2, or any combination thereof. In some embodiments, one or more (e.g.at least 1, 2, 3, 4, 5) of the Notch activators or ligands DLL1, DLL3,DLL4, JAG1, or JAG2, or any combination thereof, is provided at aconcentration that is, is about, is at least, is at least about, is notmore than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, or 50 μg/mL, or any concentration within a rangedefined by any two of the aforementioned concentrations, for example, 1ng/mL, to 50 μg/mL, 5 ng/mL to 40 μg/mL, 10 ng/mL to 30 μg/mL, 1 ng/mLto 20 ng/mL or 5 μg/mL to 20 μg/mL. In some embodiments, the foregutspheroid is contacted with one or more (e.g. at least 1, 2, 3, 4, 5) ofthe Notch activators or ligands DLL1, DLL3, DLL4, JAG1, or JAG2, or anycombination thereof, for a number of days that is, is about, is atleast, is at least about, is not more than, or is not more than about,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20days, or any number of days within a range defined by any two of theaforementioned number of days, for example, 1 to 20 days, 5 to 15 days,10 to 14 days, 1 to 15 days, or 5 to 20 days. In some embodiments, theforegut spheroid comprises a cell that expresses one or more (e.g. atleast 1, 2, 3, 4 5) Notch activators or ligands selected from the groupconsisting of DLL1, DLL3, DLL4, JAG1, or JAG2, or any combinationthereof. In some embodiments, the foregut spheroid expresses one or more(e.g. at least 1, 2, 3, 4 5) Notch activators or ligands selected fromthe group consisting of DLL1, DLL3, DLL4, JAG1, or JAG2, or anycombination thereof. In some embodiments, the Notch activator or ligandis DLL4.

Described herein are methods of producing a liver organoid that produceshematopoietic stem cells. The methods comprise culturing a foregutspheroid under conditions sufficient to differentiate the foregutspheroid into a liver organoid. In some embodiments, the foregutspheroid comprises a mesenchyme. In some embodiments, the liver organoidcomprises a CD34⁺ hernogenic endothelium population that differentiateinto hematopoietic stem cells. In some embodiments, the CD34⁺ hemogenicendothelium population originates from the mesenchyme of the foregutspheroid. In some embodiments, the foregut spheroid is obtained byculturing PSCs, iPSCs, or definitive endoderm according to any one ofthe methods described herein. In some embodiments, PSCs or iPSCs arecultured according to any one of the methods described herein for anumber of days that is, is about, is at least, is at least about, is notmore than, or is not more than about, 1, 2, 3, 4, or 5 days to obtaindefinitive endoderm. In some embodiments, PSCs or iPSCs are culturedwith Activin A or BMP4, or both, for a number of days that is, is about,is at least, is at least about, is not more than, or is not more thanabout, 1, 2, 3, 4, or 5 days to obtain definitive endoderm. In someembodiments, definitive endoderm is cultured according to any one of themethods described herein for a number of days that is, is about, is atleast, is at least about, is not more than, or is not more than about,1, 2, 3, 4, or 5 days to obtain foregut spheroids. In some embodiments,definitive endoderm is cultured with FCF4 or CHIR99021, or both, for anumber of days is, is about, is at least, is at least about, is not morethan, or is not more than about, 1, 2, 3, 4, or 5 days to obtain foregutspheroids. In some embodiments, culturing the foregut spheroid comprisescontacting the foregut spheroid with DLL4. In some embodiments, DLL4 isprovided at a concentration that is, is about, is at least, is at leastabout, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 μg/mL, or any concentrationwithin a range defined by any two of the aforementioned concentrations,for example, 1 ng/mL to 50 μg/mL, 5 ng/mL to 40 μg/mL, 10 ng/mL to 30μg/mL, 1 ng/mL to 20 ng/mL, or 5 μg/mL, to 20 μg/mL. In someembodiments, the foregut spheroid is contacted with DLL4 for a number ofdays that is, is about, is at least, is at least about, is not morethan, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 days, or any number of days within arange defined by any two of the aforementioned number of days, forexample, 1 to 20 days, 5 to 15 days, 10 to 14 days, 1 to 15 days, or 5to 20 days.

Described herein are methods of producing a liver organoid that produceshematopoietic stem cells. The methods comprise culturing a foregutspheroid under conditions sufficient to differentiate the foregutspheroid into a liver organoid. In some embodiments, the foregutspheroid comprises a mesenchyme. In some embodiments, the liver organoidcomprises a CD34⁺ hetnogenic endothelium population that differentiateinto hematopoietic stem cells. In some embodiments, the CD34⁺ hemogenicendothelium population originates from the mesenchyme of the foregutspheroid. In some embodiments, the foregut spheroid is obtained byculturing PSCs, iPSCs, or definitive endoderm according to any one ofthe methods described herein. In some embodiments, PSCs or iPSCs arecultured according to any one of the methods described herein for anumber of days that is, is about, is at least, is at least about, is notmore than, or is not more than about, 1, 2, 3, 4, or 5 days to obtaindefinitive endoderm. In some embodiments. PSCs or iPSCs are culturedwith Activin A or BMP4, or both, for a number of days that is, is about,is at least, is at least about, is not more than, or is not more thanabout, 1, 2, 3, 4, or 5 days to obtain definitive endoderm. In someembodiments, definitive endoderm is cultured according to any one of themethods described herein for a number of days that is, is about, is atleast, is at least about, is not more than, or is not more than about,1, 2, 3, 4, or 5 days to obtain foregut spheroids. In some embodiments,definitive endoderm is cultured with FCF4 or CHIR99021, or both, for anumber of days is, is about, is at least, is at least about, is not morethan, or is not more than about, 1, 2, 3, 4, or 5 days to obtain foregutspheroids. In some embodiments, culturing the foregut spheroid comprisescontacting the foregut spheroid with a cytokine. In some embodiments,the cytokine is provided at a concentration that is, is about, is atleak, is at least about, is not more than, or is not more than about, 0,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, or 200 ng/mL, or any concentration within a range definedby any two of the aforementioned concentrations, for example, 0 to 200ng/mL, 50 to 150 ng/mL, 80 to 120 ng/mL, 0 to 100 ng/mL, or 100 to 200ng/mL. In some embodiments, the cytokine is one or more (e.g. at least1, 2, 3) of IL-3, IL-34, or GM-CSF, or any combination thereof. In someembodiments, the foregut spheroid is contacted with the Notch activatoror ligand for a number of days that is, is about, is at least, is atleast about, is not more than, or is not more than about, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, or anynumber of days within a range defined by any two of the aforementionednumber of days, for example, 1 to 20 days, 5 to 15 days, 10 to 14 days,1 to 15 days, or 5 to 20 days,

Described herein are methods of producing a liver organoid that produceshematopoietic stem cells. The methods comprise culturing a foregutspheroid under conditions sufficient to differentiate the foregutspheroid into a liver organoid. In some embodiments, the foregutspheroid comprises a mesenchyme. In some embodiments, the liver organoidcomprises a CD34⁺ hemogenic endothelium population that differentiateinto hematopoietic stem cells. In some embodiments, the CD34⁺ hemogenicendothelium population originates from the mesenchyme of the foregutspheroid. In some embodiments, the foregut spheroid is obtained byculturing PSCs, iPSCs, or definitive endoderm according to any one ofthe methods described herein. In some embodiments, PSCs or iPSCs arecultured according to any one of the methods described herein for anumber of days that is, is about, is at least, is at least about, is notmore than, or is not more than about, 1, 2, 3, 4, or 5 days to obtaindefinitive endoderm. In some embodiments, PSCs or iPSCs are culturedwith Activin A or BMP4, or both, for a number of days that is, is about,is at least, is at least about, is not more than, or is not more thanabout, 1, 2, 3, 4, or 5 days to obtain definitive endoderm. In someembodiments, definitive endoderm is cultured according to any one of themethods described herein for a number of days that is, is about, is atleast, is at least about, is not more than, or is not more than about,1, 2, 3, 4, or 5 days to obtain foregut spheroids. In some embodiments,definitive endoderm is cultured with FGF4 or CHIR99021, or both, for anumber of days is, is about, is at least, is at least about, is not morethan, or is not more than about, 1, 2, 3, 4, or 5 days to obtain foregutspheroids. In some embodiments, culturing the foregut spheroid comprisescontacting the foregut spheroid with one or more (e.g. at least 1, 2, 3,4, 5) of the cytokines SCF, IL-3, IL-6, IL-34, EPO, TPO, G-CSF, or(IM-CSF, or any combination thereof. In some embodiments, one or more(e.g. at least 1, 2, 3, 4, 5) of the cytokines SCF, IL-3, IL-6, IL-34,EPO, TPO, G-CSF, or GM-CSF, or any combination thereof, is provided at aconcentration that is, is about, is at least, is at least about, is notmore than, or is not more than about, 0, 1, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng/mL, or anyconcentration within a range defined by any two of the aforementionedconcentrations, for example, 0 to 200 ng/mL, 5 to 10 ng/mL, 20 to 100ng/mL, or 100 to 200 ng/mL. In some embodiments, one or more (e.g., atleast 1, 2, 3, 4, 5) of the cytokines SCF, IL-3, IL-6, IL-34, EPO, TPO,G-CSF, or GM-CSF, or any combination thereof, is provided at aconcentration that is, is about, is at least, is at least about, is notmore than, or is not more than about, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100μg/mL, or any concentration within a range defined by any two of theaforementioned concentrations, for example, 0 to 100 μg/mL, 1 to 10μg/mL, 10 to 50 μg/mL, 20 to 100 mg/mL, or 5 to 15 mg/mL. In someembodiments, the foregut spheroid is contacted with one or more (e.g. atleast 1, 2, 3, 4, 5) of the cytokines SCF, IL-3, IL-6, IL-34, EPO, TPO,G-CSF, or GM-CSF, or any combination thereof, for a number of days thatis, is about, is at least, is at least about, is not more than, or isnot more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 days, or any number of days within a range definedby any two of the aforementioned number of days, for example, 1 to 20days, 5 to 15 days, 10 to 14 days, 1 to 15 days, or 5 to 20 days.

Described herein are methods of producing a liver organoid that produceshematopoietic stem cells. The methods comprise culturing a foregutspheroid under conditions sufficient to differentiate the foregutspheroid into a liver organoid. In some embodiments, the foregutspheroid comprises a mesenchyme. In some embodiments, the liver organoidcomprises a CD34⁺ hemogenic endothelium population that differentiateinto hematopoietic stem cells. In some embodiments, the CD34⁺ hemogenicendothelium population originates from the mesenchyme of the foregutspheroid. In some embodiments, the foregut spheroid is obtained byculturing PSCs, iPSCs, or definitive endoderm according to any one ofthe methods described herein. In some embodiments, PSCs or iPSCs arecultured according to any one of the methods described herein for anumber of days that is, is about, is at least, is at least about, is notmore than, or is not more than about, 1, 2, 3, 4, or 5 days to obtaindefinitive endoderm. In some embodiments, PSCs or iPSCs are culturedwith Activin A or BMP4, or both, for a number of days that is, is about,is at least, is at least about, is not more than, or is not more thanabout, 1, 2, 3, 4, or 5 days to obtain definitive endoderm. In someembodiments, definitive endoderm is cultured according to any one of themethods described herein for a number of days that is, is about, is atleast, is at least about, is not more than, or is not more than about,1, 2, 3, 4, or 5 days to obtain foregut spheroids. In some embodiments,definitive endoderm is cultured with FGF4 or CHIR99021, or both, for anumber of days is, is about, is at least, is at least about, is not morethan, or is not more than about, 1, 2, 3, 4, or 5 days to obtain foregutspheroids. In some embodiments, culturing the foregut spheroid comprisescontacting the foregut spheroid with one or more (e.g. at least 1, 2, 3)of IL-3, IL-34, or GM-CSF, or any combination thereof. In someembodiments, one or more (e.g. at least 1, 2, 3) of IL-3, IL-34, orGM-CSF, or any combination thereof, is provided at a concentration thatis, is about, is at least, is at least about, is not more than, or isnot more than about, 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, or 200 ng/mL, or anyconcentration within a range defined by any two of the aforementionedconcentrations, for example, 0 to 200 ng/mL, 50 to 150 ng/mL, 80 to 120ng/mL, 0 to 100 ng/mL, or 100 to 200 ng/mL. In some embodiments, thecytokine is one or more (e.g. at least 1, 2, 3) of IL-3, IL-34, orGM-CSF, or any combination thereof In some embodiments, the foregutspheroid is contacted with one or more (e.g. at least 1, 2, 3) of IL-3,1L-34, or GM-CR⁻⁷, or any combination thereof, for a number of days thatis, is about, is at least, is at least about, is not more than, or isnot more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 days, or any number of days within a range definedby any two of the aforementioned number of days, for example, 1 to 20days, 5 to 15 days, 10 to 14 days, 1 to 15 days, or 5 to 20 days.

In some embodiments, any one of the methods described herein furthercomprise culturing induced pluripotent stem cells under conditionssufficient to differentiate the induced pluripotent stem cells intodefinitive endoderm and culturing the definitive endoderm underconditions sufficient to differentiate the definitive endoderm into theforegut spheroid prior to culturing the foregut spheroid underconditions sufficient to differentiate the foregut spheroid into theliver organoid. In some embodiments, any one of the methods describedherein further comprise culturing a definitive endoderm under conditionssufficient to differentiate the definitive endoderm into the foregutspheroid prior to culturing the foregut spheroid under conditionssufficient to differentiate the foregut spheroid into the liverorganoid. In some embodiments, the conditions sufficient todifferentiate the induced pluripotent stem cells into definitiveendoderm comprises contacting the induced pluripotent stem cells withActivin A or BMP4, or both, to obtain the definitive endoderm, In someembodiments, the conditions sufficient to differentiate the definitiveendoderm into the foregut spheroid comprises contacting the definitiveendoderm with FGF4 or CHIR99021, or both, to obtain the foregutspheroid. In some embodiments, the foregut spheroid comprises mesoderm,mesoderm cells, mesodermal progenitor cells, mesenchyme, mesenchymalcells, mesenchymal stem cells, or mesenchymal progenitor cells, or anycombination thereof. In some embodiments, the foregut spheroid comprisesmesenchyme. In some embodiments, one or more of the induced pluripotentstem cells, definitive endoderm, foregut spheroids, or liver organoid,or any combination thereof is prepared according to methods describedherein. In some embodiments, one or more of the induced pluripotent stemcells, definitive endoderm, foregut spheroids, or liver organoid, or anycombination thereof is prepared according to methods described in PCTPublications WO 2018/085615, WO 2018/191673, WO 2018/226267, WO2019/126626, WO 2020/023245, WO 2020/056158, and WO 2020/069285, each ofwhich is hereby expressly incorporated by reference for the purposes ofproducing induced pluripotent stem cells, definitive endoderm, foregutspheroids, or liver organoids, or any combination thereof.

As described herein, some embodiments of liver organoids produce one ormore (e.g. at least 1, 2, 3) of hemogenic endothelium cells,hematopoietic cells, hematopoietic stem cells, or hematopoieticprogenitor cells, or any combination thereof. In some embodiments, theone or more of hemogenic endothelium cells, hematopoietic cells,hematopoietic stem cells, or hematopoietic progenitor cells, or anycombination thereof, originate from mesenchyme of the liver organoids.In some embodiments, the one or more of hemogenic endothelium cells,hematopoietic cells, hematopoietic stem cells, or hematopoieticprogenitor cells, or any combination thereof, originate from mesenchymeof the foregut spheroids that differentiate into the liver organoids. Insome embodiments, the hemogenic endothelium cells, hematopoietic cells,hematopoietic stem cells, or hematopoietic progenitor cells, or anycombination thereof, are isolated from the liver organoids. In someembodiments, the hemogenic endothelium cells, hematopoietichematopoietic stem cells, or hematopoietic progenitor cells, or anycombination thereof, that are isolated are administered to an individualin need. In some embodiments, the hemogenic endothelium cells,hematopoietic cells, hematopoietic stem cells, or hematopoieticprogenitor cells, or any combination thereof, that are isolated from theliver organoid are allogeneic to the individual. In some embodiments,the liver organoid is autologous to the individual, and as aconsequence, the hemogenic endothelium cells, hematopoietic cells,hematopoietic stem cells, or hematopoietic progenitor cells, or anycombination thereof, are also autologous to the individual. In someembodiments, the hemogenic endothelium cells, hematopoietic cells,hematopoietic stem cells, or hematopoietic progenitor cells, or anycombination thereof, are not exogenously expressing one or more (e.g. atleast 1, 3, 5) of ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, SPI1, FOSB, orGHI1, or any combination thereof.

In some embodiments, the methods described herein comprise isolating thehemogenic endothelium population or the hematopoietic stem cells, orboth, from the liver organoid. In some embodiments, isolating thehemogenic endothelium population or the hematopoietic stem cells, orboth, is performed by sorting for a cell population from the liverorganoid that is CD34+, CD45+, or both.

In some embodiments, the methods described herein comprise isolatinglymphoid cells from a liver organoid. In some embodiments, the liverorganoid is any one of the liver organoids produced by any one of themethods described herein. In some embodiments, the lymphoid cells areisolated from any one of the liver organoids produced by any one of themethods described herein. In some embodiments, the lymphoid cellscomprise one or more of B cells, T cells, NK cells, or common lymphoidprogenitor cells. In some embodiments, the B cells are B1 cells. In someembodiments, the B cells are B2 cells.

In some embodiments, the B cells are both B1 cells and B2 cells. In someembodiments, the lymphoid cells that are isolated are B1 cells. In someembodiments, the methods described herein comprise making a single cellsuspension from the liver organoid. In some embodiments, the methodsdescribed herein comprise culturing the single cell suspension onstromal cells. In some embodiments, the methods described hereincomprise culturing the single cell suspension on stromal cells prior toisolating the lymphoid cells. In some embodiments, culturing the singlecell suspension on stromal cells expands the lymphoid cells in thesingle cell suspension. In some embodiments, the stromal cells are MS-5cells. In some embodiments, the single cell suspension is cultured onstromal cells for a number of weeks that is, is about, is at least, isat least about, is not more than, or is not more than about, 2, 3, 4, or5 weeks. In some embodiments, the single cell suspension is contactedwith one or more cytokines. In some embodiments, one or more cytokines,or any combination thereof, is provided at a concentration that is, isabout, is at least, is at least about, is not more than, or is not morethan about, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, or 200 ng/mL, or any concentration within arange defined by any two of the aforementioned concentrations, forexample, 0 to 200 ng/mL, 5 to 10 ng/mL, 20 to 100 ng/mL, or 100 to 200ng/mL. In some embodiments, the liver organoid is contacted with one ormore cytokines for a number of days that is, is about, is at least, isat least about, is not more than, or is not more than about, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, orany number of days within a range defined by any two of theaforementioned number of days, for example, 1 to 20 days, 5 to 15 days,10 to 14 days, 1 to 15 days, or 5 to 20 days. In some embodiments, thesingle cell suspension is contacted with one or more (e.g. at least 1,2, 3) of SCF, IL-3, IL-6, IL-7, IL 34, EPO, TPO, G-CSF, GM-CST, orFins-like tyrosine kinase 3 ligand (FLT3 ligand, FLT3L), or anycombination thereof. In some embodiments, one or more (e.g. at least 1,2, 3) of SCF, IL-6, IL-7, IL-34, EPO, TPO, G-CSF, GM-CSF, or FLT3ligand, or any combination thereof, is provided at a concentration thatis, is about, is at least, is at least about, is not more than, or isnot more than about, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, or 200 ng/mL, or any concentration withina range defined by any two of the aforementioned concentrations, forexample, 0 to 200 ng/mL, 5 to 10 ng/mL, 20 to 100 ng/mL, or 100 to 200ng/mL. In some embodiments, one or more (e.g. at least 1, 2, 3) of SCF,IL-3, IL-6, IL-7, IL-34, EPO, TPO, G-CSF, GM-CSF, or FLT3 ligand, or anycombination thereof, is provided at a concentration that is, is about,is at least, is at least about, is not more than, or is not more thanabout, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg/mL, or any concentrationwithin a range defined by any two of the aforementioned concentrations,for example, 0 to 100 μg/mL, 1 to 10 μg/mL, 10 to 50 μg/mL, 20 to 100μg/mL, or 5 to 15 μg/mL. In some embodiments, the liver organoid iscontacted with one or more (e.g. at least 1, 2, 3) of SCF, IL-3, IL-6,IL-7, IL-34, EPO, G-CSF, GM-CSF, or FLT3 ligand, or any combinationthereof, for a number of days that is, is about, is at least, is atleast about, is not more than, or is not more than about, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, or anynumber of days within a range defined by any two of the aforementionednumber of days, for example, 1 to 20 days, 5 to 15 days, 10 to 14 days,1 to 15 days, or 5 to 20 days. In some embodiments, the single cellsuspension is contacted with one or more (e.g. at least 1, 2, 3) of FLT3ligand, SCF, or IL-7, or any combination thereof. In some embodiments,one or more (e.g. at least 1, 2, 3) of FLT3 ligand, SCF, or IL-7, or anycombination thereof, is provided at a concentration that is, is about,is at least, is at least about, is not more than, or is not more thanabout, 0, 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, or 200 ng/mL, or any concentration within a range definedby any two of the aforementioned concentrations, for example, 0 to 200ng/mL, 5 to 10 ng/mL, 20 to 100 ng/mL, or 100 to 200 ng/rriL. In someembodiments, the liver organoid is contacted with one or more (e.g. atleast 1, 2, 3) of FLT3 ligand, SCF, or IL-7, or any combination thereof,for a number of days that is, is about, is at least, is at least about,is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, or any number ofdays within a range defined by any two of the aforementioned number ofdays, for example, 1 to 20 days, 5 to 15 days, 10 to 14 days, 1 to 15days, or 5 to 20 days. In some embodiments, isolating the lymphoid cellsis performed by sorting for a cell population from the single cellsuspension. In some embodiments, the lymphoid cells are isolated fromthe single cell suspension after culturing for a number of days that is,is about, is at least, is at least about, is not more than, or is notmore than about, 10, 11 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, or 50 days, or any number of days withina range defined by any two of the aforementioned number of days, forexample, 10 to 50 days, 15 to 40 days, 20 to 30 days, 10 to 30 days, or20 to 50 days. In some embodiments, the B1 cells areCD20⁺CD43⁺CD27⁺CD70⁻. In some embodiments, isolating the B1 cells isperformed by sorting for a cell population from the single cellsuspension that is one or more (e.g. at least 1, 2, 3) of CD20⁺, CD43⁺,or CD27⁺, or any combination thereof. In some embodiments, the B1 cellshave autoimmune properties or innately express surface IgM, or both. Insome embodiments, the B1 cells are not found in UCB cells. In someembodiments, the B1 cells are isolated from the single cell suspensionafter culturing for a number of days that is, is about, is at least, isat least about, is not more than, or is not more than about, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, or 50 days, or any number of days within a range defined by any twoof the aforementioned number of days, for example, 10 to 50 days, 15 to40 days, 20 to 30 days, 10 to 30 days, or 20 to 50 days.

In some embodiments, methods of expanding a population of hematopoieticstem cells are provided. In some embodiments, the hematopoietic stemcells in the fetal liver, or liver organoid, or both, are associatedwith Nestin⁺ portal vessels. In some embodiments, the liver organoidexpresses stem cell factor (SCF) or thrombopoietin (TPO), or both, whichinduces HSC expansion and maintenance. In some embodiments, the methodcomprises culturing the population of hematopoietic stem cells incontact with a liver organoid. In some embodiments, the liver organoidcomprises a hematopoietic niche environment. In some embodiments, theliver organoid is the liver organoid produced or isolated by any one ofthe methods described herein. In some embodiments, culturing thepopulation of hematopoietic stem cells comprises contacting thepopulation of hematopoietic stem cells with one or more cytokines. Insome embodiments, culturing the population of hematopoietic stem cellscomprises contacting the population of hematopoietic stem cells with SCFor TPO, or both. In some embodiments, SCF or TPO, or both, is providedat a concentration that is, is about, is at least, is at least about, isnot more than, or is not more than about, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng/mL, or anyconcentration within a range defined by any two of the aforementionedconcentrations, for example, 0 to 200 ng/mL, 5 to 10 ng/mL, 20 to 100ng/mL, or 100 to 200 ng/mL. In some embodiments, the population ofhematopoietic stem cells is contacted with SCF or TPO, or both for anumber of days that is, is about, is at least, is at least about, is notmore than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or anynumber of days within a range defined by any two of the aforementionednumber of days, for example, 1 to 25 days, 5 to 20 days, 10 to 20 days,1 to 20 days, or 10 to 25 days. In some embodiments, the expandedpopulation of hematopoietic stem cells maintains pluripotency. In someembodiments, the expanded population of hematopoietic stem cellsmaintains pluripotency for a number of days that is, is about, is atleast, is at least about, is not more than, or is not more than about,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 days, or any number ofdays within a range defined by any two of the aforementioned number ofdays, for example, 1 to 50 days, 5 to 40 days, 10 to 30 days, 1 to 30days, or 20 to 50 days. In some embodiments, the population ofhematopoietic stem cells is obtained from umbilical cord blood,mobilized peripheral blood, bone marrow, pluripotent stem cells,embryonic stem cells, fetal liver, or fetal spleen, or any combinationthereof In some embodiments, liver organoids that are differentiatedwith one or more (e.g. at least 1, 2, 3, 4) of RA, dexamethasone,Oncostatin M, or hepatocyte growth factor, or any combination thereof,do not support hematopoietic cells as well as undifferentiated,fetal-like liver organoids.

In some embodiments, the population of hematopoietic stem cells that areexpanded are grafted into an individual. In some embodiments, theindividual is a mouse. In some embodiments, the individual is animmunocompromised mouse. In some embodiments, the individual is a human.In some embodiments, the individual is an immunocompromised human. Insome embodiments, the individual is an individual in need ofhematopoietic stem cells. In some embodiments, the population ofhematopoietic stem cells is injected into the individual. In someembodiments, the population of hematopoietic stem cells is injectedintrafemorally. In some embodiments, the population of hematopoieticstem cells is injected with liver organoid cells. In some embodiments,the population of hematopoietic stem cells is injected after isolatingthe population of hematopoietic stem cells, where there are no liverorganoid cells left. In some embodiments, the individual is alsoadministered SCF or TPO, or both. In some embodiments, the population ofhematopoietic stem cells engraft into the individual. In someembodiments, the individual in need of hematopoietic stem cells is inneed for the treatment, inhibition, or amelioration of conditionsincluding but not limited to aplastic anemia, Fanconi anemia,Diamond-Blackfan syndrome, sickle cell disease, thalassemia, paroxysmalnocturnal hemoglobinuria, Chediak-Higashi syndrome, chronicgranulomatous disease, Glanzmann thrombasthenia, osteopetrosis, Gaucherdisease, Niemann-Pick disease, mucopolysaccharidosis, glycoproteinoses,immune deficiencies, AIDS, ataxia telangiectasia, DiGeorge syndrome,severe combined immunodeficiency, Wiscott-Aldrich syndrome, Kostmannsyndrome, Shwachman-Diamond syndrome, leukemia, acute myelogenousleukemia, acute lymphoblastic leukemia, hairy cell leukemia, chroniclymphocytic leukemia, myelodysplasia, Hodgkin disease, Non-Hodgkindisease, multiple myeloma, myeloproliferative neoplasm, myelofibrosis,polycythemia vera, chronic myelogenous leukemia, neuroblastoma,desmoplastic small round cell tumor, Ewing sarcoma, or choriocarcinoma,or any combination thereof.

In some embodiments, the population of hematopoietic stem cells isautologous or allogeneic to the liver organoid. In some embodiments, thepopulation of hematopoietic stem cells is obtained from an individual.In some embodiments, the liver organoid is differentiated from cellsobtained from an individual. In some embodiments, the liver organoid isproduced from induced pluripotent stem cells derived from an individual.In some embodiments, the population of hematopoietic stem cells andliver organoid are obtained or derived from the same individual. In someembodiments, the individual is an individual in need of hematopoieticstem cells.

Described herein are methods of administering to an individual in need apopulation of hematopoietic stem cells. In some embodiments, thepopulation of hematopoietic stem cells is an isolated population ofhematopoietic stem cells. In some embodiments, the population ofhematopoietic stem cells is expanded, produced, or isolated, or anycombination thereof, according to any one of the methods describedherein. In some embodiments, the population of hematopoietic stem cellsis autologous or allogeneic to the individual in need. In someembodiments, the population of hematopoietic stem cells is autologous tothe individual in need. In some embodiments, the population ofhematopoietic stem cells is allogeneic to the individual in need.

Described herein are methods of administering to an individual in need ahemogenic endothelium population. In some embodiments, the hemogenicendothelium population is an isolated hemogenic endothelium population.In some embodiments, the hemogenic endothelium population is produced orisolated, or both, according to any one of the methods described herein.In some embodiments, the hemogenic endothelium population is autologousto the individual in need. In some embodiments, the hemogenicendothelium population is allogeneic to the individual in need.

Described herein are methods of administering to an individual in needlymphoid cells. In some embodiments, the lymphoid cells are isolatedlymphoid cells. In some embodiments, the lymphoid cells are produced orisolated, or both, according to any one of the methods described herein.In some embodiments, the lymphoid cells are autologous to the individualin need. In some embodiments, the lymphoid cells are allogeneic to theindividual in need. In some embodiments, the lymphoid cells comprise oneor more of B cells, T cells, NK cells, or common lymphoid progenitorcells, or any combination thereof. In some embodiments, the B cells areB1 cells or B2 cells, or both.

Described herein are methods of administering to an individual in needB1 cells, In some embodiments, the B1 cells are isolated B1 cells. Insome embodiments, the B1 cells are produced or isolated, or both,according to any one of the methods described herein. In someembodiments, the B1 cells are autologous to the individual in need. Insome embodiments, the B1 cells are allogeneic to the individual in need.

Described herein are embodiments of a cell composition. In someembodiments, the cell composition comprises the liver organoid producedby any one of the methods described herein. In some embodiments, thecell composition comprises the hemogenic endothelium population producedor isolated by any one of the methods described herein. In someembodiments, the cell composition comprises the hematopoietic stem cellsproduced or isolated by any one of the methods described herein. In someembodiments, the cell composition comprises the B1 cells produced orisolated by any one of the methods described herein. In someembodiments, the cell composition comprises any combination of the othercell compositions described herein. In some embodiments, the cellcomposition comprises one or more (e.g. at least 1, 2, 3, 4) of theliver organoid, hernogenic endothelium population, hematopoietic stemcells, or B1 cells, or any combination thereof disclosed herein. In someembodiments, the cell composition comprises a population ofhematopoietic stem cells and a liver organoid. In some embodiments, thepopulation of hematopoietic stem cells is prepared or isolated, or both,according to any one of the methods described herein. In someembodiments, the liver organoid is prepared or isolated, or both,according to any one of the methods described herein. In someembodiments, the population of hetnatopoietic stem cells and liverorganoid are from, obtained from, or derived from the same individual.In some embodiments, the population of hematopoietic stem cells andliver organoid are from, obtained from, or derived from differentindividuals.

EXAMPLES

Some aspects of the embodiments discussed above are disclosed in furtherdetail in the following examples, which are not in any way intended tolimit the scope of the present disclosure. Those in the art willappreciate that many other embodiments also fall within the scope of thedisclosure, as it is described herein above and in the claims.

Example 1. Material and Methods hPSC Maintenance

Human iPSC lines were maintained as described previously (Takebe et al.,2015; Takebe et al. 2014). Undifferentiated hiPSCs were maintained onfeeder-free conditions in StemFit medium (StemCell Technologies) onplates coated with Laminin (1% iMatrix-511 silk in PBS) for 2 hours at37° C. iPSCs were seeded at 8×10⁴ cells/well of 6-well cell culturetreated plates (BD Falcon) in StemFit media supplemented with 100 ng/mLFGF2 and 10 nM ROCK inhibitor Y-27632 at 37° C. in 5% CO₂. cl DefinitiveEndoderm Induction

Differentiation of human iPSCs into definitive endoderm was inducedusing previously described methods with slight modifications (Spence etal. 2011). Briefly, colonies of human iPSCs were isolated in Accutase(Thermo Fisher) in Dulbecco's phosphate buffered saline (DPBS, LifeTechnologies) and 150,000 cells/mL were plated on Matrigel of a 1/30dilution on tissue culture plates in RPMI 1640 medium (LifeTechnologies) supplemented with 100 ng/mL Activin A (R&D Systems) and 50ng/mL bone morphogenic protein 4 (BMPI, R&D Systems) at day 1, and 100ng/mL Activin A and 2% fetal calf serum (FCS) at day 3. On days 4-6,cells were cultured in Advanced DMFNI (Thermo Fisher) with B27 (LifeTechnologies) and N2 (Gibco) supplements and additionally supplementedwith 500 ng/int, fibroblast growth factor 4 (FGF4, R&D Systems) and 3 μMCHIR99021 (Stemgent). Cells were maintained at 37° C. in 5% CO₂, and themedium was replaced every day. Spheroids appeared on the plate at day 7of differentiation.

Human Liver Organoid (HLO) Formation

Day 7 spheroids were collected by manual pipetting and pelleted bycentrifugation. Media was aspirated and cells were embedded in 100%Matrigel by gentle pipetting to homogenize the cell pellet. Optionally,a Notch activator or ligand is added to the Matrigel (e.g. 10 μg/mL, ofDLIA). Alternatively, optionally, UCB cells are added to the Matrigelfor co-culture with organoids (e.g. 1,000 UCB cells per 75 μL ofMatrigel). 250 μL Matrigel (Corning) was used per well of a 24-wellplate of definitive endoderm culture. 75 μL Matrigel drops were made, 1drop per each well of a fresh 24-well plate (VWR). The plates wereplaced at 37° C. in an atmosphere of 5% CO₂/95% air for 5-15 minutes.After the Matrigel was solidified, Advanced DMEM/F12 with 1×B27, 1×N2, 2mM L-glutamine, 15 mM HEPES, and 1× penicillin/streptomycin was added tothe wells. This media was supplemented with 20 ng/mL BMP4 and 10 ng/mL,FGF2 for 4 days, and then the BMP4 and FGF2 were removed.

Albumin ELISA

To measure albumin secretion level of HLOs, 200 μL of culturesupernatant was collected from HLOs embedded in Matrigel. The culturesupernatants were collected 24 hours after media changes and stored at−80° C. until use. The supernatant was assayed with a Human AlbuminELISA Quantitation Set kit (Bethyl Laboratories, E80-129) according tomanufacturer's instructions.

Histology

Samples in Matrigel drops were collected, fixed in 4% paraformaldehydeand embedded in paraffin. Sections were subjected to hematoxylin andeosin (H&E) and immunohistochemical staining. The following primaryantibodies were used: anti-human albumin (Sigma. Aldrich, A668),anti-human CD34 (Roche, 760-2620), anti-human CD45 (Roche, 760-2505).

Flow Cytometry

Organoids were isolated from Matrigel drops and washed with 1×PBS. HLOswere dissociated to single cells by treatment with Trypsin-EDTA (0.05%,with phenol red, Gibco) for 10 minutes. Cells were washed in DMEM/F12with 10% FBS and suspended in PBS with 2% FBS. After filtration (e.g.through a 40-50 μm nylon filter), the single cells were subjected toflow cytometry with the following antibodies: anti-CD45-APC (Miltenyl,130-098-143), anti-CD34-APC/Cy7 (BioLegend, 343513), anti-CD11b-PE/Cy7(BioLegend, 301322), anti-CD19-BV421 (BioLegend, 302233),anti-CD27-AF700 (BioLegend, 356415), anti-CD43-FITC (BioLegend, 315204).For mouse engraftment experiments, anti-mouse CD45-BV421 (BioLegend,103133) was used.

Colony Forming Cell (CFC) Assay

150,000 cells/mL were plated into 3 mL of MethoCult SF H4436 serum-freemethylcellulose-based medium (StemCell Technologies). The mixture wasevenly distributed into 60 mm dishes and maintained in a humidifiedchamber at 37° C. for 14 days followed by examination by lightmicroscopy. The media used contains insulin, transferrin, SCF, IL3,IL-6, EPO, G-CSF, and GM-CSF as cytokines. SCF, IL-3, IL-6, G-CSF, andGM-CSF are supplemented at 100 ng/mL, EPO is supplemented at 1-8 IU/mL(approximately 10-100 ng/mL).

Cytospin and Giemsa Stain

Cells from CFC assays were collected from methylcellulose media bywashing with PBS and 7,000 cells were applied as a thin layer on glassslides using a Cytospin centrifuge (500 rpm for 10 minutes). Slides wereair dried and stained with a Wright-Giemsa staining kit (Thermo Fisher)according to manufacturer's instructions, followed by examination bylight microscopy.

Mouse Transplantation

NSG-SGM3 (NSGS, NOD-scid IL2Rgnull-3/GM/SF) mice were bred and housed inan animal care facility. Animal experiments were performed in accordancewith institutional guidelines approved by an animal care committee. Micewere sub-lethally immune ablated with Busulfan (30 μg/g of body weightin 10 μL volume) administered by IP injection 24 hours prior to cellinjection. Cells were intrafemorally injected. Organoid cultures wereco-cultured with umbilical cord blood (UCB) CD34⁺ cells and medium wassupplemented with stem cell factor (SCF) and thrombopoietin (TPO) for 12days. 2×10⁶ HLO+HUCB cells or 2×10³ UCB CD34⁺ cells only were injectedinto mice. After 7 weeks, femoral bone marrow and blood were collectedand analyzed by flow cytometry.

RNA Isolation and RT-qPCR

RNA was isolated using the RNeasy mini kit (Qiagen). Reversetranscription was carried out using the High-Capacity cDNA. ReverseTranscription Kit (Thermo Fisher) according to manufacturer's protocol.qPCR was carried out in duplicate using TagMan Gene Expression MasterMix (Applied Biosystems) and probes from the Universal Probe Library(Roche) on a QuantStudio 3 Real-Time PCR System (Thermo Fisher). Allprimer and probe information for each target gene was obtained from theUniversal Probe Library Assay Design Center (accessible on the worldwide web at qpcr.probefinder.com/organism.jsp). The primers are asfollows:

Alpha-fetoprotein (AFP) Forward: (SEQ ID NO: 1)tgtactgcagagataagtttagctgac AFP Reverse: (SEQ ID NO: 2)tccttgtaagtggcttcttgaac Albumin (ALB) Forward: (SEQ ID NO: 3)gtgaggttgctcatcggttt ALB Reverse: (SEQ ID NO: 4) gagcaaaggcaatcaacaccCD34 Forward: (SEQ ID NO: 5) ggcacagctggaggtcttat CD34 Reverse:(SEQ ID NO: 6) tgttcaggcatcagagaagtgHemoglobin subunit gamma-1 (HBGI) Forward: (SEQ ID NO: 7)tggatcctgagaacttcaagc HBG1 Reverse: (SEQ ID NO: 8) gccactgcagtcaccatctHES1 Forward: (SEQ ID NO: 9) gaagcacctccggaacct HES1 Reverse:(SEQ ID NO: 10) gtcacctcgttcatgcactc HES5 Forward: (SEQ ID NO: 11)cccggggttctatgatatttg HES5 Reverse: (SEQ ID NO: 12)gccctgaagaaagtcctctaca

Accession numbers are available as HUGO Gene Nomenclature Committee Ms:AFP (HGNC: 317), ALB (HGNC: 399), CD34 (HGNC: 1662), HBG1(HGNC: 4831),HES1 (HGNC: 5192), HES5 (HGNC: 19764), EPO (HGNC: 3415), TPO (THPO,HGNC:11795), SCF (KIT ligand, KITLG, HGNC:6343).

Single Cell RNA Sequencing

HLOs were isolated from Matrigel drops and washed with 1×PBS. HLOs weredissociated to single cells by treatment with Trypsin-EDTA (0.05%, withphenol red, Gibco) for 10 minutes. Cells were washed in DMEM/F12 with10% FBS and suspended in PBS with 2% EBS. Cells then underwent singlecell RNA-sect library prep and were sequenced with the 10× GenomicsChromium platform and 2868 cells were recovered for analysis. Sequencedreads were processed using the Cell Ranger gene expression pipelinesmkfastq and count, starting with demultiplexing and conversion ofbarcode and read data to fastq files. Raw reads were aligned to the Hg19genome and filtered, creating gene-barcode matrices. Analysis wereperformed in AltAnalyze, where gene and cell clusters were identified byunsupervised analysis to identify predominant sample groups viaexpression clustering. Following removal of outliers, clusters wererestricted to having >5 cells, a minimum Pearson correlation of 0.5, anda between-cluster fold change >4. Cell cycle effects were removed.Cluster-based heatmaps and corresponding tSNE plots were generated usingRv3.3.3. Cell cluster identity was determined through lineagecorrelation and ontological analysis in ToppGene (available on the worldwide web at toppgene.cchmc.org). The tSNE plot was established by LoupeCell Browser v1.0.5.

Example 2. Parallel Differentiation of Hepatic Endoderm and HemogenicEndothelium

In order to mimic the fetal liver environment, iPSC-derived midgutspheroids were produced through endoderm induction in the presence ofActivin A and embedded in Matrigel drops as described herein. Midgutspheroids were then cultured in the presence of BMP4 and FGF2, which areimportant in early hepatic specification. The resulting organoidcultures expressed hepatic genes, including albumin (ALB) andalpha-fetoprotein (AFP), and the endothelial marker CD34 (FIGS. 1A-D).The organoids also comprised a hematopoietic population that expressedfetal hemoglobin (as evidenced by the presence of hemoglobin gammasubunits). Additional analysis revealed that the organoid systemcontained a hemogenic endothelial population that was capable ofproducing erythrocytes without the addition of exogenous hematopoieticcytokines (FIG. 1A). This was further verified by single cell RNAsequencing (scRNA-seq) of day 14 organoid cultures. Expression profileanalysis showed that at this time point, there were three populations ofinterest, in addition to a large mesenchyme population expressingmesoderm and ectoderm markers (FIGS. 2, 3). The clusters of interestcomprised a hepatic population, an erythroid population, and a hemogenicendothelium population (FIGS. 4A-C). These hemogenic endothelial cellswere further analyzed through colony forming cell (CFC) assays, in whichcells from organoid cultures were dispersed into a single cellsuspension and plated onto methylcellulose-based media with cytokines.The plated cells produced both erythroid and myeloid colonies, with apeak colony formation occurring around organoid culture day 14 (FIG.5A-B).

Example 3. Emergence of Notch Dependent Hemogenic Endothelium

Histological examination of liver organoid cultures revealed epithelialcell adhesion molecule (EpCAM)-expressing cells alongside CD34⁺endothelium with adjacent erythroid cells expressing glycophorin A(CD235a, GYPA) as well as CD45 expressing hematopoietic cells (FIG.6A-B). Time course analysis of CD34-expressing cells revealed that thenumber of CD34⁺ cells decreased prior to a peak in CFC assay activity(FIG. 6C-D).

Notch signaling has previously been shown to be important inestablishing definitive hematopoiesis in hemogenic endothelium.Specifically, Notch receptors Notch1 and Notch4, along with Notchligands JAG1, JAG2, and DLL4 are expressed by aortic endothelial cellsand are essential for early hematopoiesis through hemogenic endothelium.During this process, endothelial cells expressing the receptor Notchl orNotch4 can interact with adjacent endothelial cells expressing theligand. DLL4 leading to increased expression of downstream target genessuch as HES1/5, HEY1/2, and GATA2. A sharp increase in Notch signalingis evidenced by increased HES1 expression levels that peak around day 10of organoid culture. This peak is immediately prior to the peak inhemogenic activity demonstrated by CFC assays, which peak around day 12to 14 (FIGS. 6D-E), Flow cytometry of organoid cells to detect CD34showed a steady decrease in CD34⁺ cell numbers as organoidsdifferentiated (FIG. 6C). To better understand the population dynamicsof the organoid systems described herein, day 14 organoid cultures wereanalyzed by scRNA-seq. Closer analysis of the CD34⁺ endothelialpopulation revealed very specific expression of Notch signaling genesNotch 1, Notch4, HEY1, HEY2, DLL4, and GATA2 (FIGS. 7A, 4C).

To test NotCh dependency in the organoid culture system, the Notchligand DLL4 (1-10 μg/mL) was added to the Matrigel drops during organoidseeding, and the organoid growth medium was supplemented withhematopoietic cytokines IL-3, IL-34, and GM-CSF (100 ng/mL each) (FIG.7B). Addition of DLL4 increased the efficiency of cytokines in producingCD45⁺ hematopoietic cells (FIG. 8). This resulted in a significantincrease in IL-6 expression with only a modest decrease in albuminexpression (FIGS. 9A-B). Conversely, organoid cultures treated with the₇-secretase inhibitor DAPT to inhibit Notch activity resulted in adecrease in visible erythrocytes and HES1 expression while maintainingalbumin expression (FIGS. 10A-D).

Example 4, Creation of B1 Cells from Organoid. Cultures

Definitive hematopoiesis is characterized by the production of bothmyeloid and lymphoid lineages. In order to examine the lymphoidpotential of the organoid cultures described herein, cells werecollected around organoid culture day 14 and plated onto confluent MS-5murine stromal cells to differentiate into B cells (FIGS. 11A-B). MS-5cells are cultured in media supplemented with 5 ng/mL FLT3 ligand(FLT3L), 100 ng/mL SCF, and 5 ng/mL 1L-7. During development, the fetalliver is a source of the unique B1 cell population. These cells aredifferent from adult (B2) cells in that they arise independently fromHSCs. B1 cells have autoimmune properties, innately express surface IgM,and have been described to be CD20⁺CD43⁺CD27⁺CD70⁻. Cells from organoidswere compared to UCB cells in their production of B cells from MS-5co-cultures. Interestingly, while both organoids and UCB were able toexhibit B cell differentiation, organoid-derived. B cell populationsincluded a unique B1 cell phenotype that was not able to be recreatedfrom UCB cells. FACS analysis revealed a population of CD20⁺CD43⁺CD27⁺cells (FIG. 11C).

Example 5. Creation of a Hematopoietic Niche Environment

Hematopoietic stem and progenitor cells exist within a specific andcomplex niche environment that is not yet fully understood, particularlyduring fetal development during which HSCs in the fetal liver undergomassive expansion. In order to better understand the origin of thehematopoietic cells in the organoid cultures described herein, organoidcells were analyzed for hematopoietic niche markers. HSCs in the fetalliver are associated with Nestin⁺ portal vessels. Within the organoidcultures, pockets of CD34⁺ cells were also seen in close associationwith Nestin staining, and scRNA-seq also showed a population ofCD34/Nestin co-expressing cells (FIGS. 12A-B, 4A-D). Interestingly,scRNA-seq also revealed that cells clustered with hepatic populationsexpressed stem cell factor (SCF) and thrombopoietin (TPO) (FIG. 12C),which are essential for HSC expansion and maintenance and are often usedin attempts to expand HSCs ex vivo.

To further test the niche environment in organoid cultures, CD34⁺ humanUCB cells were added to the Matrigel drops during organoid seeding.Exogenous recombinant SCE and TPO was added to the growth medium toimprove the efficiency of hematopoietic cell production and maintenance.Addition of 100 ng/mL SCF and 10 ng/mL TPO resulted in a significantincrease in CD34^(+ and CD)45⁺ cell production in the organoid culturesas well as an increase in the number of colonies formed (FIGS. 12D-E).

To determine if these results were due to the presence of ahematopoietic niche or merely a contaminating mesoderm populationresulting in hematopoietic differentiation, the ability of UCB cells toform colonies after mixing with the organoid cultures was examined.Promotion of hepatic differentiation of the organoids with the additionof retinoic acid (RA) followed by hepatocyte culture medium (HCM,Lonza), optionally supplemented with 0.1 μM dexamethasone, 10 ng/inLOncostatin M, and 10 ng/triL hepatocyte growth factor (HGF), resulted ina steady decline of hematopoietic colony forming ability. Conversely,the protocols described herein modified to limit hepatic differentiationmaintains colony forming cell presence as well as their diversedifferentiation potential, demonstrating the existence of a unique nichethat models the transient stage of fetal liver hematopoiesis and relieson the presence of an immature hepatic population (FIGS. 12F-G).

UCB cells are capable of engrafting into immunocotnpromised recipientsand re-establishing the complete hematopoietic system. However, alimiting factor in the potential therapeutic application of these cellsis their inability to be cultured or expanded in vitro, In order tofurther analyze the niche environment of the organoid systems describedherein, the engraftment of UCB cells after co-culture with organoids wastested. UCB cells were combined with organoids in Matrigel drops and thegrowth medium was supplemented with SCF and TPU at organoid culture day6. After 12 days, UCB and organoid cells were collected into a singlecell suspension and intrafemorally injected into the bone marrow of NSGSmice, Seven weeks later, mice showed engraftment of human specific CD45⁺cells (FIGS. 13A-B). The resulting engraftment was around 3% engraftmentafter co-culturing the UCB cells with organoids compared to around 16%engraftment without co-culture. Although this engraftment is decreasedcompared to fresh cord blood cells, UCB cultured in the same conditionswithout organoids immediately lost their engraftment potential. Thus,this provides a new system for culturing HSC while maintainingengraftment capability.

Shown herein, a novel fetal liver organoid system derived from humaniPSCs is capable of giving rise to both hepatic and hematopoietic cellsin a symbiotic co-differentiation system. Hematopoietic cells in thissystem arise from a hemogenic endothelium population and differentiateinto both myeloid and lymphoid lineages in the presence of cytokines.Co-culture of organoid cells with MS-5 mouse stromal cells give rise toa fetal Bl-like cell population as identified by flow cytometry.Although there was only limited engraftment after co-culture of UCBcells with the organoids, this system provides evidence for a novel wayto maintain HSC in culture through mimicking their natural environment.

In at least some of the previously described embodiments, one or moreelements used in an embodiment can interchangeably be used in anotherembodiment unless such a replacement is not technically feasible. Itwill be appreciated by those skilled in the art that various otheromissions, additions and modifications may be made to the methods andstructures described herein without departing from the scope of theclaimed subject matter. All such modifications and changes are intendedto fall within the scope of the subject matter, as defined by theappended claims.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein fbr sakeof clarity.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “a”system having “at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible sub-rangesand combinations of sub-ranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into sub-ranges as discussed herein. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 articles refers to groupshaving 1, 2, or 3 articles. Similarly, a group having 1-5 articlesrefers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

All references cited herein, including but not limited to published andunpublished applications, patents, and literature references, areincorporated herein by reference in their entirety and are hereby made apart of this specification. To the extent publications and patents orpatent applications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

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Lis R, Karrasch C C, Poulos M G, et al. Conversion of adult endotheliumto itntnunocompetent haematopoietic stem cells. Nature. 2017; 545(7655):439-445. doi: 10. 1038/nature22326

Wilkinson A C, Ishida R, Kikuchi M, et al. Long-term ex vivohaematopoietic-stem-cell expansion allows nonconditionedtransplantation. Nature. 2019;571(7763)117-121. doi:10,1038/s41586-019-1244-x

Rothstein T L, Griffin D O, Holodick N E, Quach T D, Kaku H. Human B-1cells take the stage. Ann N Y Acad Sci. 2013;1285:97-114.doi:10.1111/nyas.12137

Montecino-Rodriguez E, Leathers H, Dorshkind K. Identification of a B-1B cell-specified progenitor. Nat Inimunol. 2006; 7(3):293-301. doi:10.1038/nil301

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What is claimed is:
 1. A method of producing a liver organoid thatproduces hematopoietic stem cells (HSCs), comprising: culturing aforegut spheroid under conditions sufficient to differentiate theforegut spheroid into a liver organoid; wherein the liver organoidcomprises a CD34⁺ hemogenic endothelium population that differentiateinto hematopoietic stem cells; and wherein the foregut spheroidcomprises mesenchyme.
 2. The method of claim 1, wherein culturing theforegut spheroid further comprises contacting the foregut spheroid witha Notch activator or ligand,
 3. The method of claim 2, wherein the Notchactivator or ligand comprises one or more of DLL1, DLL3, DLL4, JAG1, orJAG2, or any combination thereof.
 4. The method of claim 2 or 3, whereinthe Notch activator or ligand comprises a cell that expresses one ormore of DLL1, DLL3, DLL4, JAG1, or JAG2, or any combination thereof. 5.The method of any one of the preceding claims, wherein the foregutspheroid further comprises a cell that expresses one or more Notchactivators or ligands selected from the group consisting of DLL1, DLL3,DLL4, JAG1, or JAG2, or any combination thereof.
 6. The method of anyone of the preceding claims, wherein the foregut spheroid expresses oneor more Notch activators or ligands selected from the group consistingof DIL1, DLL3, JAG1, or JAG2, or any combination thereof.
 7. The methodof any one of the preceding claims, wherein culturing the foregutspheroid further comprises contacting the foregut spheroid with one ormore of IL-3, IL-34, or GM-CSF, or any combination thereof.
 8. Themethod of any one of the preceding claims, wherein culturing the foregutspheroid further comprises contacting the foregut spheroid with stemcell factor (SCF) or thrombopoietin (TPO), or both.
 9. The method of anyone of the preceding claims, wherein culturing the foregut spheroid doesnot comprise contacting the foregut spheroid with retinoic acid (RA).10. The method of any one of the preceding claims, wherein culturing theforegut spheroid does not comprise contacting the foregut spheroid withone or more of dexamethasone, Oncostatin M, or hepatocyte growth factor,or any combination thereof.
 11. The method of any one of the precedingclaims, wherein the hetnatopoietic stem cells are not exogenouslyexpressing one or more of ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, SPI1,FOSB, or GFI1, or any combination thereof,
 12. The method of any one ofthe preceding claims, further comprising isolating the hemogenicendothelium population or the hematopoietic stem cells, or both, fromthe liver organoid.
 13. The method of claim 12, wherein isolating thehen:log⁻eine endothelium population or the hematopoietic stem cells, orboth, is performed by sorting for a cell population from the liverorganoid that is CD34⁺, CD45⁺, or both.
 14. The method of any one of thepreceding claims, further comprising: culturing induced pluripotent stemcells under conditions sufficient to differentiate the inducedpluripotent stem cells into definitive endoderm; and culturing thedefinitive endoderm under conditions sufficient to differentiate thedefinitive endoderm into the foregut spheroid; prior to culturing theforegut spheroid under conditions sufficient to differentiate theforegut spheroid into the liver organoid.
 15. The liver organoidproduced by the method of any of the preceding claims.
 16. The homogenicendothelium population produced by the method of any of the precedingclaims.
 17. The hematopoietic stem cells produced by the method of anyof the preceding claims.
 18. The method of any one of claims 1-14,further comprising isolating lymphoid cells from the liver organoid. 19.The method of claim 18, wherein the lymphoid cells comprise one or moreof B cells, T cells, NK cells, or common lymphoid progenitor cells. 20.The method of claim 19, wherein the B cells are B1 cells or B2 cells, orboth.
 21. The method of any one of claims 18-20, further comprisingmaking a single cell suspension from the liver organoid and culturingthe single cell suspension on stromal cells prior to isolating thelymphoid cells, wherein culturing the single cell suspension on stromalcells expands lymphoid cells in the single cell suspension.
 22. Themethod of claim 21, wherein the stromal cells are MS-5 cells.
 23. Themethod of claim 21 or 22, wherein the single cell suspension iscontacted with one or more of FLT3 ligand. SCF, or IL-7, or anycombination thereof.
 24. The method of any one of claims 20-23, whereinthe B1 cells are CD20⁺TD43⁺CD27⁺ and isolating the B1 cells is performedby sorting for a cell population from the single cell suspension that isone or more of CD20^(±), CD43′, CD27±, or any combination thereof. 25.The lymphoid cells produced by the method of any one of claims 18-24.26. The B1 cells produced by the method of any one of claims 20-24. 27.A method of expanding a population of hematopoietic stem cells,comprising culturing the population of hematopoietic stem cells incontact with a liver organoid, wherein the liver organoid comprises ahematopoietic niche environment.
 28. The method of claim 27, wherein theliver organoid is the liver organoid produced by the method of any oneof the preceding claims.
 29. The method of claim 27 or 28, whereinculturing the population of hematopoietic stem cells further comprisescontacting the population of hematopoietic stem cells with SCF or TPO,or both.
 30. The method of any one of claims 27-29, wherein the expandedpopulation of hematopoietic stem cells maintain pluripotency.
 31. Themethod of any one of claims 27-30, wherein the population ofhematopoietic stem cells is obtained from umbilical cord blood,mobilized peripheral blood, bone marrow, pluripotent stem cells,embryonic stem cells, fetal liver, or fetal spleen, or any combinationthereof.
 32. The method of any one of claims 27-31, wherein thepopulation of hematopoietic stem cells is autologous or allogeneic tothe liver organoid.
 33. The method of any one of claims 27-32, whereinthe population of hematopoietic stem cells is obtained from anindividual.
 34. The method of claim 33, wherein the individual is anindividual in need of hematopoietic stem cells.
 35. The method of claim33 or 34, wherein the liver organoid is produced from inducedpluripotent stem cells derived from the individual.
 36. A methodcomprising administering to an individual in need the population ofhematopoietic stem cells expanded according to the method of any one ofclaims 27-35.
 37. The method of claim 36, wherein the population ofhematopoietic stem cells is autologous or allogeneic to the individualin need.
 38. A cell composition comprising a population of hematopoieticstem cells and a liver organoid.
 39. A cell composition comprising apopulation of hematopoietic stem cells and a liver organoid, wherein thepopulation of hematopoietic stem cells are produced by the method of anyone of claims 27-35.
 40. A cell composition comprising a population ofhematopoietic stem cells and a liver organoid, wherein the liverorganoid is produced by the method of any one of claims 1-14.
 41. Thecell composition of any one of claims 38-40, wherein the population ofhematopoietic stem cells and liver organoid are from the sameindividual.
 42. The cell composition of any one of claims 38-40, whereinthe population of hematopoietic stem cells and liver organoid are fromdifferent individuals.
 43. A method comprising administering to anindividual in need the isolated hemogenic endothelium population ofclaim 16 or the isolated hematopoietic stem cells of claim
 17. 44. Amethod comprising administering to an individual in need the isolatedlymphoid cells of claim
 25. 45. A method comprising administering to anindividual in need the isolated B1 cells of claim 26.